Systems, devices, and methods for visualization of tissue and collection and analysis of data regarding same

ABSTRACT

A system for determining a bacterial load of a target is provided. The system includes an adaptor for configuring a mobile communication device for tissue imaging and a mobile communication device. The adaptor includes a housing configured to be removably coupled to a mobile communication device and, an excitation light source for fluorescent imaging. The excitation light source is configured to emit light in one of ultraviolet, visible, near-infrared, and infrared ranges.

This application is a continuation of U.S. application Ser. No.16/593,174, filed Oct. 4, 2019, and issued as U.S. Pat. No. 11,676,276on Jun. 13, 2023, which is a continuation of U.S. application Ser. No.15/328,214, filed Jan. 23, 2017, which is a U.S. national stageapplication under 35 U.S.C. § 371(c) of International Application No.PCT/CA2015/000444, filed on Jul. 24, 2015, which claims benefit of U.S.Provisional Application No. 62/028,386, filed Jul. 24, 2014, the entirecontents of each of which are incorporated by reference herein.

This application is a continuation of U.S. application Ser. No.18/148,861, filed Dec. 30, 2022, which is a continuation of U.S.application Ser. No. 16/593,174, filed Oct. 4, 2019, and issued as U.S.Pat. No. 11,676,276 on Jun. 13, 2023, which is a continuation of U.S.application Ser. No. 15/328,214, filed Jan. 23, 2017, which is a U.S.national stage application under 35 U.S.C. § 371(c) of InternationalApplication No. PCT/CA2015/000444, filed on Jul. 24, 2015, which claimsbenefit of U.S. Provisional Application No. 62/028,386, filed Jul. 24,2014, the entire contents of each of which are incorporated by referenceherein.

TECHNICAL FIELD

Devices and methods for collecting data for diagnostic purposes aredisclosed. In particular, the devices and methods of the presentapplication may be suitable for evaluating and tracking bacterial loadin a wound over time.

BACKGROUND

Wound care is a major clinical challenge. Healing and chronicnon-healing wounds are associated with a number of biological tissuechanges including inflammation, proliferation, remodeling of connectivetissues and, a common major concern, bacterial infection. A proportionof wound infections are not clinically apparent and contribute to thegrowing economic burden associated with wound care, especially in agingpopulations. Currently, the gold-standard wound assessment includesdirect visual inspection of the wound site under white light combinedwith indiscriminate collection of bacterial swabs and tissue biopsiesresulting in delayed, costly and often insensitive bacteriologicalresults. This may affect the timing and effectiveness of treatment.Qualitative and subjective visual assessment only provides a gross viewof the wound site, but does not provide information about underlyingbiological and molecular changes that are occurring at the tissue andcellular level. A relatively simple and complementary method thatcollects and analyzes ‘biological and molecular’ information inreal-time to provide early identification of such occult change andguidance regarding treatment of the same is desirable in clinical woundmanagement. Early recognition of high-risk wounds may guide therapeuticintervention and provide response monitoring over time, thus greatlyreducing both morbidity and mortality due especially to chronic wounds.

SUMMARY

In accordance with one aspect of the present disclosure, a system fordetermining a bacterial load of a target is provided. The systemcomprises an adaptor for configuring a mobile communication device forfluorescent imaging of a target and a mobile communication device. Theadaptor comprises a housing configured to be removably coupled to amobile communication device, and an excitation light source configuredto emit excitation light selected to elicit emission of bacterialautofluorescence from bacteria in a target illuminated with theexcitation light. The mobile communication device comprises an opticalsensor configured to detect signals responsive to illumination of thetarget with the excitation light, and a processor. The processor isconfigured to receive the signals responsive to illumination of thetarget with the excitation light and corresponding to bacterialautofluorescence of the target, to analyze the signals using pixelintensity, and to output data regarding a bacterial load of the target.

Additional objects and advantages of the disclosure will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the disclosure. Theobjects and advantages of the disclosure will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the disclosure andtogether with the description, serve to explain the principles of thedisclosure.

BRIEF DESCRIPTION OF DRAWINGS

At least some features and advantages will be apparent from thefollowing detailed description of embodiments consistent therewith,which description should be considered with reference to theaccompanying drawings, wherein:

FIG. 1 is a schematic diagram of an exemplary device forfluorescence-based monitoring in accordance with the present disclosure;

FIG. 2A shows an exemplary charging stand for use with a device forfluorescence-based monitoring in accordance with the present disclosure;

FIG. 2B shows an example of a clinical wound care facility using adevice for fluorescence-based monitoring in accordance with the presentdisclosure;

FIG. 3 shows images of a muscle surface of a pig meat sample,demonstrating the exemplary use of a device for fluorescence-basedmonitoring in accordance with the present disclosure forautofluorescence detection of connective tissues and bacteria;

FIG. 4 shows images of an exemplary handheld embodiment of a device forfluorescence-based monitoring in accordance with the present disclosure;

FIGS. 5A and 5B show alternate exemplary embodiments of a handhelddevice for obtaining white light and fluorescent light data from atarget in accordance with the present disclosure;

FIGS. 6A-6D show alternative exemplary embodiments of a handheld devicefor obtaining data regarding a target, the handheld device incorporatingan iPhone;

FIGS. 7A and 7B illustrate exemplary methods of determining bacterialload of a target in accordance with the present disclosure;

FIG. 8 shows representative white light (WL) and fluorescent (FL) imagesfor a single mouse tracked over 10 days;

FIG. 9 illustrates preclinical data which show that pathogenic bacterialautofluorescence (AF) intensity correlates with bacterial load in vivo;

FIG. 10 shows images of live bacterial cultures captured using a devicefor fluorescence-based monitoring in accordance with the presentdisclosure;

FIG. 11 shows an example of bacterial monitoring using a device forfluorescence-based monitoring in accordance with the present disclosure;

FIG. 12 shows images of a simulated animal wound model, demonstratingnon-invasive autofluorescence detection of bacteria using a device forfluorescence-based monitoring in accordance with the present disclosure;

FIG. 13 illustrates an example of monitoring of a chronic wound;

FIGS. 14-28 show examples of the use of a device for fluorescence-basedmonitoring in accordance with the present disclosure for imaging woundsand conditions in clinical patients;

FIG. 29 shows images of a skin surface of a pig meat sample,demonstrating non-invasive autofluorescence detection of collagen andvarious bacterial species using a device for fluorescence-basedmonitoring in accordance with the present disclosure;

FIG. 30 shows images and spectral plots demonstrating the use of adevice for fluorescence-based monitoring in accordance with the presentdisclosure to detect fluorescence from bacteria growing in agar platesand on the surface of a simulated wound on pig meat;

FIG. 31 shows images demonstrating use of a device forfluorescence-based monitoring in accordance with the present disclosurefor imaging of blood and microvasculature;

FIG. 32 is a flowchart illustrating the management of a chronic woundusing a device for fluorescence-based monitoring in accordance with thepresent disclosure;

FIG. 33 illustrates the phases of wound healing with time;

FIG. 34 is a table showing examples of tissue, cellular and molecularbiomarkers known to be associated with wound healing;

FIG. 35 is a diagram comparing a healthy wound to a chronic wound;

FIG. 36 shows images demonstrating the use of a device forfluorescence-based monitoring in accordance with the present disclosurein imaging a mouse model;

FIG. 37 shows an example of the use of a device for fluorescence-basedmonitoring in accordance with the present disclosure for imaging smallanimal models;

FIG. 38 shows an example of an exemplary kit including a device forfluorescence-based monitoring in accordance with the present disclosure;

FIG. 39 shows an example of the use of a device for fluorescence-basedmonitoring in accordance with the present disclosure for imaging a skinsurface;

FIG. 40 shows images demonstrating additional exemplary uses of a devicefor fluorescence-based monitoring in accordance with the presentdisclosure for imaging a skin surface;

FIG. 41 shows an example of the use of a device for fluorescence-basedmonitoring in accordance with the present disclosure for imagingcosmetic or dermatological substances; and

FIG. 42 shows an example of the use of a device for fluorescence-basedmonitoring in accordance with the present disclosure with an exemplarydrape.

Although the following detailed description makes reference toillustrative embodiments, many alternatives, modifications, andvariations thereof will be apparent to those skilled in the art.Accordingly, it is intended that the claimed subject matter be viewedbroadly.

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments, examples ofwhich are illustrated in the accompanying drawings. The variousexemplary embodiments are not intended to limit the disclosure. To thecontrary, the disclosure is intended to cover alternatives,modifications, and equivalents.

Conventional clinical assessment methods of acute and chronic woundscontinue to be suboptimal. Such assessment methods usually are based ona complete patient history, qualitative and subjective clinicalassessment with simple visual appraisal using ambient white light andthe ‘naked eye,’ and can sometimes involve the use of color photographyto capture the general appearance of a wound under white lightillumination. Regular re-assessment of progress toward healing andappropriate modification of the intervention is also necessary. Woundassessment terminology is non-uniform, many questions surrounding woundassessment remain unanswered, agreement has yet to be reached on the keywound parameters to measure in clinical practice, and the accuracy andreliability of available wound assessment techniques vary.

Visual assessment is frequently combined with swabbing and/or tissuebiopsies for bacteriological culture for diagnosis. Bacterial swabs arecollected at the time of wound examination and have the noted advantageof providing identification of specific bacterial/microbial species.However, multiple swabs and/or biopsies often are collected randomlyfrom the wound site, and some swabbing techniques may in fact spread themicroorganisms around with the wound during the collection process thusaffecting patient healing time and morbidity. This may be a problemespecially with large chronic (non-healing) wounds where the detectionyield for bacterial presence using current swabbing and biopsy protocolsis suboptimal (diagnostically insensitive), despite many swabs beingcollected.

Thus, current methods for obtaining swabs or tissue biopsies from thewound site for subsequent bacteriological culture are based on anon-targeted or ‘blind’ swabbing or punch biopsy approach, and have notbeen optimized to minimize trauma to the wound or to maximize thediagnostic yield of the bacteriology tests. In addition, bacteriologicalculture results often take about 2-3 days to come back from thelaboratory and can be inconclusive, thus delaying accurate diagnosis andtreatment. Thus, conventional methods of obtaining bacterial swabs donot necessarily provide relevant data regarding the wound and cannotprovide real-time detection of infectious status of wounds. The lack ofa non-invasive method to objectively and rapidly evaluate wound repairat a biological level (which may be at greater detail than simplyappearance or morphology based), and to aid in targeting of thecollection of swab and tissue biopsy samples for bacteriology is a majorobstacle in clinical wound assessment and treatment. An alternativemethod is highly desirable.

As wounds (chronic and acute) heal, a number of key biological changesoccur at the wound site at the tissue and cellular level. Wound healinginvolves a complex and dynamic interaction of biological processesdivided into four overlapping phases—haemostasis, inflammation, cellularproliferation, and maturation or remodeling of connective tissues—whichaffect the pathophysiology of wound healing. A common major complicationarising during the wound healing process, which can range from days tomonths, is infection caused by bacteria and other microorganisms. Thiscan result in a serious impediment to the healing process and lead tosignificant complications. All wounds contain bacteria at levels rangingfrom contamination, through colonization, critical colonization toinfection, and diagnosis of bacterial infection is based on clinicalsymptoms and signs (e.g., visual and odorous cues).

The most commonly used terms for wound infection have included woundcontamination, wound colonisation, wound infection and, more recently,critical colonisation. Wound contamination refers to the presence ofbacteria within a wound without any host reaction; wound colonisationrefers to the presence of bacteria within the wound which do multiply orinitiate a host reaction; and critical colonisation refers tomultiplication of bacteria causing a delay in wound healing, usuallyassociated with an exacerbation of pain not previously reported butstill with no overt host reaction. Wound infection refers to thedeposition and multiplication of bacteria in tissue with an associatedhost reaction. In practice the term ‘critical colonisation’ can be usedto describe wounds that are considered to be moving from colonisation tolocal infection. The challenge within the clinical setting, however, isto ensure that this situation is quickly recognized with confidence andfor the bacterial bioburden to be reduced as soon as possible, perhapsthrough the use of topical antimicrobials. Potential wound pathogens canbe categorised into different groups, such as, bacteria, fungi, spores,protozoa and viruses depending on their structure and metaboliccapabilities. Although viruses do not generally cause wound infections,bacteria can infect skin lesions formed during the course of certainviral diseases. Such infections can occur in several settings includingin health-care settings (hospitals, clinics) and at home or chronic carefacilities. The control of wound infections is increasingly complicated,yet treatment is not always guided by microbiological diagnosis. Thediversity of micro-organisms and the high incidence of polymicrobicflora in most chronic and acute wounds give credence to the value ofidentifying one or more bacterial pathogens from wound cultures. Theearly recognition of causative agents of wound infections can assistwound care practitioners in taking appropriate measures. Furthermore,faulty collagen formation arises from increased bacterial burden andresults in over-vascularized friable loose granulation tissue thatusually leads to wound breakdown.

Accurate and clinically relevant wound assessment is an importantclinical tool, but this process currently remains a substantialchallenge. Current visual assessment in clinical practice only providesa gross view of the wound site (e.g., presence of purulent material andcrusting). Current best clinical practice fails to adequately use thecritically important objective information about underlying keybiological changes that are occurring at the tissue and cellular level(e.g., contamination, colonization, infection, matrix remodeling,inflammation, bacterial/microbial infection, and necrosis) since suchindices are i) not easily available at the time of the wound examinationand ii) they are not currently integrated into the conventional woundmanagement process. Direct visual assessment of wound health statususing white light relies on detection of color andtopographical/textural changes in and around the wound, and thus may beincapable and unreliable in detecting subtle changes in tissueremodeling. More importantly, direct visual assessment of wounds oftenfails to detect the presence of bacterial infection, since bacteria areoccult under white light illumination. Infection is diagnosed clinicallywith microbiological tests used to identify organisms and theirantibiotic susceptibility. Although the physical indications ofbacterial infection can be readily observed in most wounds using whitelight (e.g., purulent exudate, crusting, swelling, erythema), this isoften significantly delayed, and the patient is already at increasedrisk of morbidity (and other complications associated with infection)and mortality. Therefore, standard white light direct visualizationfails to detect the early presence of the bacteria themselves oridentify the types of bacteria within the wound.

Wound progression is currently monitored manually. The National PressureUlcer Advisory Panel (NPUAP) developed the Pressure Ulcer Scale forHealing (PUSH) tool that outlines a five-step method of characterizingpressure ulcers. This tool uses three parameters to determine aquantitative score that is then used to monitor the pressure ulcer overtime. The qualitative parameters include wound dimensions, tissue type,and the amount of exudate or discharge, and thermal readings presentafter the dressing is removed. A wound can be further characterized byits odor and color. Such an assessment of wounds currently does notinclude critical biological and molecular information about the wound.Therefore, all descriptions of wounds are somewhat subjective and notedby hand by either the attending physician or the nurse.

What is desirable is a robust, cost-effective non-invasive and rapidimaging-based method or device for collecting wound data and providingan analysis in real-time. The data and analysis can be used toobjectively assess wounds for changes at the biological, biochemical andcellular levels and to rapidly, sensitively and non-invasively detectingthe earliest presence of bacteria/microorganisms within wounds. Such amethod or device for detection of critical biological tissue changes inwounds may serve an adjunctive role with conventional clinical woundmanagement methods in order to guide key clinico-pathological decisionsin patient care. Such a device may be compact, portable and capable ofreal-time non-invasive and/or non-contact interrogation of wounds in asafe and convenient manner, which may allow it to fit seamlessly intoroutine wound management practice and user friendly to the clinician,nurse and wound specialist. This may also include use of this device inthe home-care environment (including self-use by a patient), as well asin military battlefield environments. In addition, such an image-baseddevice may provide an ability to monitor wound treatment response andhealing in real-time by incorporating valuable ‘biologically-informed’image-guidance into the clinical wound assessment process. This mayultimately lead to potential new diagnosis, treatment planning,treatment response monitoring and thus ‘adaptive’ interventionstrategies which may permit enhancement of wound-healing response at theindividual patient level. Precise identification of the systemic, local,and molecular factors underlying the wound healing problem in individualpatients may allow better tailored treatment.

In accordance with the present teachings, methods of analysis for datacollected from a wound are provided. For example, the collection offluorescence image data appears to be promising for improving clinicalwound assessment and management. When excited by short wavelength light(e.g., ultraviolet or short visible wavelengths), most endogenousbiological components of tissues (e.g., connective tissues such collagenand elastins, metabolic co-enzymes, proteins, etc.) produce fluorescenceof a longer wavelength, in the ultraviolet, visible, near-infrared andinfrared wavelength ranges.

Tissue autofluorescence imaging provides a unique means of obtainingbiologically relevant information of normal and diseased tissues inreal-time, thus allowing differentiation between normal and diseasedtissue states, as well as the volume of the diseased tissue. This isbased, in part, on the inherently different light-tissue interactions(e.g., abosption and scattering of light) that occur at the bulk tissueand cellular levels, changes in the tissue morphology and alterations inthe blood content of the tissues. In tissues, blood is a major lightabsorbing tissue component (i.e., a chromophore). This type oftechnology is suited for imaging disease in hollow organs (e.g., GItract, oral cavity, lungs, bladder) or exposed tissue surfaces (e.g.,skin). An autofluorescence imaging device in accordance with the presentdisclosure may collect wound data that provides/allows rapid,non-invasive and non-contact real-time analysis of wounds and theircomposition and components, to detect and exploit the rich biologicalinformation of the wound to improve clinical care and management.

A device in accordance with the present disclosure: 1) providesimage-guidance for tissue sampling, detecting clinically-significantlevels of pathogenic bacteria and wound infection otherwise overlookedby conventional sampling and 2) provides image-guidance for woundtreatment, accelerating wound closure compared with conventionaltherapies and quantitatively tracking long-term changes in bacterialbioburden and distribution in wounds.

U.S. Pat. No. 9,042,967 B2 to DaCosta et al., entitled “Device andMethod for Wound Imaging and Monitoring,” and issued on May 26, 2015,discloses at least some aspects of a device configured to collect datafor objectively assessing wounds for changes at the biological,biochemical and cellular levels and for rapidly, sensitively andnon-invasively detecting the earliest presence ofbacteria/microorganisms within wounds. This patent claims priority toPCT Application No. PCT/CA2009/000680 filed on May 2009, and to U.S.Provisional Patent Application No. 61/054,780, filed on May 20, 2008.The entire content of each of these above-identified patents, patentapplications, and patent application publications is incorporated hereinby reference.

In accordance with one aspect of the present teachings, a handheldportable device to examine skin and wounds in real-time is provided. Thedevice instantly detects, visualizes, and analyzes bacteria and tissuecomposition. The device is a compact, handheld, device for noncontactand noninvasive imaging. It captures both white light (WL) andautofluorescence (AF) signals produced by tissue components and bacteriawithout the use of contrast agents. Although capable of detecting AFsignals without use of contrast agents, one of ordinary skill in the artwill understand that the devices disclosed herein can be used withcontrast agents if desired. In addition to white light and fluorescence,the device also may capture thermal data from the imaged area. Thedevice may be further configured to analyze the white light,fluorescence, and thermal data, correlate such data, and provide anoutput based on the correlation of the data, such as, for example, anindication of wound status, wound healing, wound infection, bacterialload, or other diagnostic information upon which an interventionstrategy may be based.

The device may be configured to create and/or display composite imagesincluding green AF, produced by endogenous connective tissues (e.g.,collagen, elastin) in skin, and red AF, produced by endogenousporphyrins in clinically relevant bacteria such as Staphylococcusaureus. Siderophores/pyoverdins in other species such as Pseudomonasaeruginosa appear blue-green in color with in vivo AF imaging. Thedevice may provide visualization of bacterial presence, types,distribution, amounts in and around a wound as well as key informationsurrounding tissue composition (collagen, tissue viability, blood oxygensaturation). For example, the device may provide imaging of collagencomposition in and around skin in real-time (via AF imaging).

In accordance with various exemplary embodiments of the presentdisclosure, the device may be configured to accurately detect andmeasure bacterial load in wounds in real-time, guide treatmentdecisions, and track wound healing over the course of antibacterialtreatment. Additionally, bioluminescence imaging (BLI) may be used tocorrelate absolute bacterial load with FL signals obtained using thehandheld device. The device may produce a uniform illumination field ona target area to allow for imagining/quantification of bacteria,collagen, tissue viability, and oxygen saturation.

The device may produce high-quality, focused images. The device mayinclude software that provides macro zoom correction, auto-focus, autowhite balance, wide dynamic range, noise reduction, image stabilization,and FL image calibration. In some embodiments, the device operates at anambient temperature between about 0-35° C.

The device may be independent and self-contained. It may interface withcomputers, printers and EMR systems.

In accordance with one exemplary embodiment of the present disclosure,the device is configured to image bacteria in real-time (via, forexample, fluorescence imaging), permitting ready identification ofbacteria types, their location, distribution and quantity in acceptedunits of measurement and allowing identification of and distinctionbetween several different species of bacteria. For example,autofluorescence imaging may be used to visualize and differentiatePseudomonas aruginosa (which fluoresces a greenish-blue colour whenexcited by 405 nm light from the device) from other bacteria (e.g.,Staphylococcus aureus) that predominantly fluoresce a red/orange colourunder the same excitation wavelength. In one exemplary embodiment thedevice's camera sensor and built in fluorescence multiband pass emissionfilter produce fluorescence images of bacteria (in wounds or normalskin) and Pseudomonas aruginosa appear greenish-blue in colour whileother bacteria emit a red/orange colour. The device detects differencesin the autofluorescence emission of different endogenous molecules(called fluorophores) between the different bacteria.

In accordance with another exemplary embodiment of the presentdisclosure, the device is configured to identify or provide anindication of tissue viability in real-time (via fluorescence imaging).For example, blood preferentially absorbs 405 nm light compared withother visible wavelengths. Tissues which are perfused by blood areconsidered viable and can be differentiated from devitalized (poorlyperfused) tissues using fluorescence imaging. Using 405 nm light from adevice in accordance with the present teachings to illuminate a wound,the device can be configured with a multiband pass emission filter todetect the amount of 405 nm light that is absorbed or reflected from thetissues. Viable tissue contains blood that highly absorbs 405 nm lightresulting in an image with low levels of 405 nm light, whereas nonviable(or devitalized) tissues do not contain sufficient blood and 405 nm isless absorbed. Thus, in an image of a wound where viable and nonviabletissues are present, the user will recognize viable tissues (fromnonviable tissues) based on the relative amount of 405 nm light in theimage, the viable tissues appearing darker compared with the nonviabletissues. In addition, in the green fluorescence “channel” of theresultant image (of the wound), viable tissues will appear less greenfluorescent compared with nonviable tissues because viable tissues willpreferentially absorb more of the 405 nm excitation light due to moreblood being present, compared with nonviable tissues. Thus, while bothviable and nonviable tissues in a resultant image obtained by the devicemay contain similar amounts of green fluorescent connective tissues(i.e., collagens), viable tissue will have less 405 nm excitation lightto stimulate the connective tissue autofluorescence than nonviabletissues. The result is that viable tissues will have less greenconnective tissue fluorescence than non-viable tissues in the sameimage. The user will appreciate this difference visually during imagingwith the device.

In accordance with another aspect of the present disclosure, the deviceis configured to capture and generate images and videos that provide amap or other visual display of user selected parameters. Such maps ordisplays may correlate, overlay, co-register or otherwise coordinatedata generated by the device based on input from one or more devicesensors. Such sensors may include, for example, camera sensorsconfigured to detect white light and/or fluorescent images and thermalsensors configured to detect heat signatures of a target. For example,the device may be configured to display color images, image maps, orother maps of user selected parameters such as, for example, bacterialocation and/or biodistribution, collagen location, location anddifferentiation between live tissues and dead tissues, differentiationbetween bacterial species, location and extent of blood, bone, exudate,temperature and wound area/size. These maps or displays may be output bythe device based on the received signals and may be produced on a singleimage with or without quantification displays. The user-selectedparameters shown on the map may be correlated with one or more woundparameters, such as shape, size, topography, volume, depth, and area ofthe wound. For example, in accordance with one exemplary embodiment, itis possible to use a ‘pseudo-coloured’ display of the fluorescenceimages/videos of wounds to color-code bacteria fluorescence (one colour)and connective tissues (another colour) etc. This may be accomplishedby, for example, using a pixel-by-pixel coloring based on the relativeamount of 405 nm light in the Blue channel of the resultant RGB image,green connective tissue fluorescence in the Green channel, and redbacteria fluorescence in Red channel. Additionally and/or alternatively,this may be accomplished by displaying the number of pixels in a givenimage for each of the blue, green and red channels which would representamount of blood in tissue, amount of connective tissues, and amount ofbacteria, respectively.

In accordance with one aspect of the present disclosure, the device maybe configured to create and output reports regarding the collected data.For example, in accordance with one exemplary embodiment, the deviceuser can generate a wound status report, which may include, for example,date/time, patient ID, images, etc. The user can export or print images,to a selected network, computer, printer when connected to cradle,and/or via USB to computer. The reports may be generated by the handhelddevice, by exporting data to a computer for processing and generation ofreports, or by a combination of the two. Further, such reports, or thedata contained therein, may form the basis of recommended interventionor treatment strategies. Reports may include, for example, medicalreports, digital reports, reports that encompass handwritten input fromclinicians (e.g., via tablet input, etc.). The reports may includevarious types of data including, for example, the identification ofwound parameters and the tracking of these parameters over time. Forexample, the reports may identify and track changes in wound size, woundshape, wound topography, wound volume, wound area, bacterial load of thewound, location of bacteria within the wound, presence of exposed bone,blood, connective and other tissues, wound temperature, location of thewound on the patient, number of wounds on the patient, date of woundexamination, patient identification, medications administered to thepatient, interventional strategies and therapies as administered and aschanged over time in response to changing wound parameters, etc. Forexample, the device may generate a report that tracks a patient's woundand skin status changes, including for example, wound size and bacterialburden over time. Further, the data collected may be used to generate adatabase that provides clinical data regarding wound parameters and theefficacy of various wound intervention/treatment strategies.Additionally, the device may be configured to integrate collecteddata/images/videos into the reports and, alternatively or additionally,include such reports and data/images/videos into a patient's electronicmedical record (EMR). This process may be wirelessly, via the use oftransfer cables, and the system also may be configured to upload thereports automatically.

The device has a memory sufficient to store several images/videos. Inaddition to internal memory, the device may include a Micro SD cardinterface for additional storage and firmware development. The devicecan inform the user of low memory capacity. The device may also includea data safeguard that will prompt a user to export files in the case oflow memory availability.

In accordance with one aspect of the present disclosure, a method anddevice for fluorescence-based imaging and monitoring is disclosed. Oneexemplary embodiment of the device is a portable optical digital imagingdevice. The device may utilize a combination of white light, tissuefluorescence and reflectance imaging, and thermal imaging, and mayprovide real-time wound imaging, assessment, recordation/documentation,monitoring and/or care management. The device may be handheld, compactand/or light-weight. This device and method may be suitable formonitoring of wounds in humans and animals.

The device may generally comprise: i) one or moreexcitation/illumination light sources and ii) a detector device (e.g., adigital imaging detector device), which may be combined with one or moreoptical emission filters, or spectral filtering mechanisms, and whichmay have a view/control screen (e.g., a touch-sensitive screen), imagecapture and zoom controls. The device may also have: iii) a wired and/orwireless data transfer port/module, iv) an electrical power source andpower/control switches, and/or v) an enclosure, which may be compactand/or light weight, and which may have a mechanism for attachment ofthe detector device and/or a handle grip. The excitation/illuminationlight sources may be LED arrays emitting light at about 405 nm (e.g.,+/−5 nm), and may be coupled with additional band-pass filters centeredat about 405 nm to remove/minimize the side spectral bands of light fromthe LED array output so as not to cause light leakage into the imagingdetector with its own optical filters. The digital imaging detectordevice may be a digital camera, for example having at least an ISO800sensitivity, but more preferably an ISO3200 sensitivity, and may becombined with one or more optical emission filters, or other equallyeffective (e.g., miniaturized) mechanized spectral filtering mechanisms(e.g., acousto-optical tunable filter or liquid crystal tunable filter).The digital imaging detector device may have a touch-sensitive viewingand/or control screen, image capture and zoom controls. The enclosuremay be an outer hard plastic or polymer shell, enclosing the digitalimaging detector device, with buttons such that all necessary devicecontrols may be accessed easily and manipulated by the user. Miniatureheat sinks or small mechanical fans, or other heat dissipating devicesmay be embedded in the device to allow excess heat to be removed fromthe excitation light sources if required. The complete device, includingall its embedded accessories and attachments, may be powered usingstandard AC/DC power and/or by rechargeable battery pack. As discussedfurther below, the battery pack may be recharged with a charging stand.

The complete device may also be attached or mounted to an externalmechanical apparatus (e.g., tripod, or movable stand with pivoting arm)allowing mobility of the device within a clinical room with hands-freeoperation of the device. Alternatively, the device may be provided witha mobile frame such that it is portable. The device may be cleaned usingmoist gauze wet with water, while the handle may be cleansed with moistgauze wet with alcohol. Additional appropriate cleaning methods will beapparent to those of ordinary skill in the art. The device may includesoftware allowing a user to control the device, including control ofimaging parameters, visualization of images, storage of image data anduser information, transfer of images and/or associated data, and/orrelevant image analysis (e.g., diagnostic algorithms). The device mayalso include one or more buttons/switches allowing a user to switchbetween white light and fluorescent light imaging.

A schematic diagram of an example of the device is shown in FIG. 1 . Thedevice is shown positioned to image a target object 10 or targetsurface. In the example shown, the device has a digital imageacquisition device 1, such as digital camera, video recorder, camcorder,cellular telephone with built-in digital camera, ‘Smart’ phone with adigital camera, personal digital assistant (PDA), laptop/PC with adigital camera, or a webcam. The digital image acquisition device 1 hasa lens 2, which may be aligned to point at the target object 10 and maydetect the optical signal that emanates from the object 10 or surface.The device has an optical filter holder 3 which may accommodate one ormore optical filters 4. Each optical filter 4 may have differentdiscrete spectral bandwidths and may be band-pass filters. These opticalfilters 4 may be selected and moved in from of the digital camera lensto selectively detect specific optical signals based on the wavelengthof light. The device may include light sources 5 that produce excitationlight to illuminate the object 10 in order to elicit an optical signal(e.g., fluorescence) to be imaged with, for example, blue light (e.g.,400-450 nm), or any other combination of single or multiple wavelengths(e.g., wavelengths in the ultraviolet/visible/near infrared/infraredranges). Thus, the device may have multiple excitation wavelengths, forexample two—three excitation wavelengths, for multiplexed fluorescence.The device also may rapidly pulse between excitation wavelengths. Thelight source 5 may comprise a LED array, laser diode and/or filteredlights arranged in a variety of geometries. The device may include amethod or apparatus 6 (e.g., a heatsink or a cooling fan) to dissipateheat and cool the illumination light sources 5. The device may include amethod or apparatus 7 (e.g., an optical band-pass filter) to remove anyundesirable wavelengths of light from the light sources 5 used toilluminate the object 10 being imaged. The device may include a methodor apparatus 8 to use an optical means (e.g., use of compact miniaturelaser diodes that emit a collimated light beam) to measure and determinethe distance between the imaging device and the object 10. For example,the device may use two light sources, such as two laser diodes, as partof a triangulation apparatus to maintain a constant distance between thedevice and the object 10. Other light sources may be possible. Thedevice may also use ultrasound, or a physical measure, such as a ruler,to determine a constant distance to maintain. In accordance with anotherexemplary embodiment, the device may use a rangefinder to determine theappropriate position of the device relative to the wound to be imaged.The device may also include a method or apparatus 9 (e.g., a pivot) topermit the manipulation and orientation of the excitation light sources5, 8 so as to manoeuvre these sources 5,8 to change the illuminationangle of the light striking the object 10 for varying distances.

The target object 10 may be marked with a mark 11 to allow for multipleimages to be taken of the object and then being co-registered foranalysis. The mark 11 may involve, for example, the use of exogenousfluorescence dyes of different colours which may produce multipledistinct optical signals when illuminated by the light sources 5 and bedetectable within the image of the object 10 and thus may permitorientation of multiple images (e.g., taken over time) of the sameregion of interest by co-registering the different colours and thedistances between them. The digital image acquisition device 1 mayinclude one or more of: an interface 12 for a head-mounted display; aninterface 13 for an external printer; an interface 14 for a tabletcomputer, laptop computer, desk top computer or other computer device;an interface 15 for the device to permit wired or wireless transfer ofimaging data to a remote site or another device; an interface 16 for aglobal positioning system (GPS) device; an interface 17 for a deviceallowing the use of extra memory; and an interface 18 for a microphone.

The device may include a power supply 19 such as an AC/DC power supply,a compact battery bank, or a rechargeable battery pack. Alternatively,the device may be adapted for connecting to an external power supply.The device may have a housing 20 that houses all the components in oneentity. The housing 20 may be equipped with a means of securing anydigital imaging device within it. The housing 20 may be designed to behandheld, compact, and/or portable. The housing 20 may be one or moreenclosures. The housing 20 may be comprised of a rugged material so thatthe device is tough and resistant to inadvertent drops by a user.Additionally, the housing 20 may include covers for any external portsof the device.

In accordance with one exemplary embodiment, the device may be chargedwhile it is stationed in a charging stand, such as charging stand 30shown in FIG. 2A. The charging stand 30 may include, for example, one ormore arms 35 to receive and securely hold the device. Additionally, thecharging stand 30 may be attached to a docking port 37, which mayinclude a cable 38 that is plugged into an outlet for charging thedevice and/or recharging a battery pack in the device. The device,charging stand 30, and/or docking port 37 may be stored in a case, suchas case 40 shown in FIG. 2A.

FIG. 2B shows an example of a device in accordance with the presentdisclosure in a typical wound care facility. Inset a) shows a typicalclinical wound care facility, showing the examination chair andaccessory table. Insets b-c) show an example of the device in itshard-case container, similar to the case 40 as shown in FIG. 2A. Thedevice may be integrated into the facility's routine wound care practiceallowing real-time imaging of a patient. Inset d) shows an example ofthe device (arrow) placed on the “wound care cart” to illustrate thesize of the device. Inset e) shows that the device may be used to imageunder white light illumination, while inset f) shows the device beingused to take fluorescence images of a wound under dimmed room lights.Inset g) shows that the device may be used in telemedicine/telehealthinfrastructures, for example fluorescence images of a patient's woundsmay be sent by email to a wound care specialist at another hospital, viaa wireless communication device, such as a Smartphone, using awireless/WiFi internet connection. Using this device, high-resolutionfluorescence images may be sent as email attachments to wound carespecialists from remote wound care sites for immediate consultation withclinical experts, microbiologists, etc. at specialized clinical woundcare and management centers.

An example of a device for fluorescence-based monitoring in accordancewith the present disclosure is described below. All examples areprovided for the purpose of illustration only and are not intended to belimiting. Parameters such as wavelengths, dimensions, and incubationtime described in the examples may be approximate and are provided asexamples only.

In this example, the device uses two violet/blue light (e.g., 405nm+/−10 nm emission, narrow emission spectrum) LED arrays (Opto DiodeCorporation, Newbury Park, California), each situated on either side ofthe imaging detector assembly as the excitation or illumination lightsources. These arrays have an output power of approximately 1 Watt each,emanating from a 2.5×2.5 cm², with a 70-degree illuminating beam angle.The LED arrays may be used to illuminate the tissue surface from adistance of about 10 cm, which means that the total optical powerdensity on the skin surface is about 0.08 W/cm². At such low powers,there is no known potential harm to either the target wound or skinsurface, or the eyes from the excitation light. However, it may beinadvisable to point the light directly at any individual's eyes duringimaging procedures. It should also be noted that 405 nm light does notpose a risk to health according to international standards formulated bythe International Electrotechnical Commission (IEC), as further detailedon the website:

-   -   http://www.iec.ch/online        news/etech/arch_2006/etech_0906/focus.htm

The one or more light sources may be articulated (e.g., manually) tovary the illumination angle and spot size on the imaged surface, forexample by using a built-in pivot, and are powered for example throughan electrical connection to a wall outlet and/or a separate portablerechargeable battery pack. Excitation/illumination light may be producedby sources including, but not limited to, individual or multiplelight-emitting diodes (LEDs) in any arrangement including in ring orarray formats, wavelength-filtered light bulbs, or lasers. Selectedsingle and multiple excitation/illumination light sources with specificwavelength characteristics in the ultraviolet (UV), visible (VIS),far-red, near infrared (NIR) and infrared (IR) ranges may also be used,and may be composed of a LED array, organic LED, laser diode, orfiltered lights arranged in a variety of geometries.Excitation/illumination light sources may be ‘tuned’ to allow the lightintensity emanating from the device to be adjusted while imaging. Thelight intensity may be variable. The LED arrays may be attached toindividual cooling fans or heat sinks to dissipate heat produced duringtheir operation. The LED arrays may emit narrow 405 nm light, which maybe spectrally filtered using a commercially available band-pass filter(Chroma Technology Corp, Rockingham, VT, USA) to reduce potential‘leakage’ of emitted light into the detector optics. When the device isheld above a tissue surface (e.g., a wound) to be imaged, theilluminating light sources may shine a narrow-bandwidth orbroad-bandwidth violet/blue wavelength or other wavelength or wavelengthband of light onto the tissue/wound surface thereby producing a flat andhomogeneous field within the region-of-interest. The light may alsoilluminate or excite the tissue down to a certain shallow depth. Thisexcitation/illumination light interacts with the normal and diseasedtissues and may cause an optical signal (e.g., absorption, fluorescenceand/or reflectance) to be generated within the tissue.

By changing the excitation and emission wavelengths accordingly, theimaging device may interrogate tissue components (e.g., connectivetissues and bacteria in a wound) at the surface and at certain depthswithin the tissue (e.g., a wound). For example, by changing fromviolet/blue (˜400-500 nm) to green (˜500-540 nm) wavelength light,excitation of deeper tissue/bacterial fluorescent sources may beachieved, for example in a wound. Similarly, by detecting longerwavelengths, fluorescence emission from tissue and/or bacterial sourcesdeeper in the tissue may be detected at the tissue surface. For woundassessment, the ability to interrogate surface and/or sub-surfacefluorescence may be useful, for example in detection and potentialidentification of bacterial contamination, colonization, criticalcolonization and/or infection, which may occur at the surface and oftenat depth within a wound (e.g., in chronic non-healing wounds). In oneexample, referring to FIG. 3 , inset c) shows the detection of bacteriabelow the skin surface (i.e., at depth) after wound cleaning. This useof the device for detecting bacteria at the surface and at depth withina wound and surrounding tissue may be assessed in the context of otherclinical signs and symptoms used conventionally in wound care centers.

Example embodiments of the device are shown in FIG. 4 . The device maybe used with any standard compact digital imaging device (e.g., acharge-coupled device (CCD) or complementary metal-oxide-semiconductor(CMOS) sensors) as the image acquisition device. The example deviceshown in a) has an external electrical power source, the two LED arraysfor illuminating the object/surface to be imaged, and a commerciallyavailable digital camera securely fixed to light-weight metal frameequipped with a convenient handle for imaging. A multi-band filter isheld in front of the digital camera to allow wavelength filtering of thedetected optical signal emanating from the object/surface being imaged.The camera's video/USB output cables allow transfer of imaging data to acomputer for storage and subsequent analysis. This example uses acommercially-available 8.1-megapixel Sony digital camera (Sony CybershotDSC-T200 Digital Camera, Sony Corporation, North America). This cameramay be suitable because of i) its slim vertical design which may beeasily integrated into the enclosure frame, ii) its large 3.5-inchwidescreen touch-panel LCD for ease of control, iii) its Carl Zeiss 5×optical zoom lens, and iv) its use in low light (e.g., ISO 3200). Thedevice may have a built-in flash which allows for standard white lightimaging (e.g., high-definition still or video with sound recordingoutput). Camera interface ports may support both wired (e.g., USB) orwireless (e.g., Bluetooth, WiFi, and similar modalities) data transferor 3 r d party add-on modules to a variety of external devices, such as:a head-mounted display, an external printer, a tablet computer, laptopcomputer, personal desk top computer, a wireless device to permittransfer of imaging data to a remote site/other device, a globalpositioning system (GPS) device, a device allowing the use of extramemory, and a microphone. The digital camera is powered by rechargeablebatteries, or AC/DC powered supply. The digital imaging device mayinclude, but is not limited to, digital cameras, webcams, digital SLRcameras, camcorders/video recorders, cellular telephones with embeddeddigital cameras, Smartphones™, personal digital assistants (PDAs), andlaptop computers/tablet PCs, or personal desk-top computers, all ofwhich contain/or are connected to a digital imaging detector/sensor.

This light signal produced by the excitation/illumination light sourcesmay be detected by the imaging device using optical filter(s) (e.g.,those available from Chroma Technology Corp, Rockingham, VT, USA) thatreject the excitation light but allow selected wavelengths of emittedlight from the tissue to be detected, thus forming an image on thedisplay. There is an optical filter holder attached to the enclosureframe in front of the digital camera lens which may accommodate one ormore optical filters with different discrete spectral bandwidths, asshown in insets b) and c) of FIG. 4 . Inset b) shows the device with theLED arrays turned on to emit bright violet/blue light, with a singleemission filter in place. Inset c) shows the device using amultiple-optical filter holder used to select the appropriate filter fordesired wavelength-specific imaging. Inset d) shows the device beingheld in one hand while imaging the skin surface of a foot.

These band-pass filters may be selected and aligned in front of thedigital camera lens to selectively detect specific optical signals fromthe tissue/wound surface based on the wavelength of light desired.Spectral filtering of the detected optical signal (e.g., absorption,fluorescence, reflectance) may also be achieved, for example, using aliquid crystal tunable filter (LCTF), or an acousto-optic tunable filter(AOTF) which is a solid-state electronically tunable spectral band-passfilter. Spectral filtering may also involve the use of continuousvariable filters, and/or manual band-pass optical filters. These devicesmay be placed in front of the imaging detector to produce multispectral,hyperspectral, and/or wavelength-selective imaging of tissues.

The device may be modified by using optical or variably orientedpolarization filters (e.g., linear or circular combined with the use ofoptical wave plates) attached in a reasonable manner to theexcitation/illumination light sources and the imaging detector device.In this way, the device may be used to image the tissue surface withpolarized light illumination and non-polarized light detection or viceversa, or polarized light illumination and polarized light detection,with either white light reflectance and/or fluorescence imaging. Thismay permit imaging of wounds with minimized specular reflections (e.g.,glare from white light imaging), as well as enable imaging offluorescence polarization and/or anisotropy-dependent changes inconnective tissues (e.g., collagens and elastin) within the wound andsurrounding normal tissues. This may yield useful information about thespatial orientation and organization of connective tissue fibersassociated with wound remodeling during healing.

All components of the imaging device may be integrated into a singlestructure, such as an ergonomically designed enclosed structure with ahandle, allowing it to be comfortably held with one or both hands. Thedevice may also be provided without any handle. The device may be lightweight, portable, and may enable real-time digital imaging (e.g., stilland/or video) of any target surface (for example, the skin and/or oralcavity, which is also accessible) using white light, fluorescence and/orreflectance imaging modes. The device may be scanned across the bodysurface for imaging by holding it at variable distances from thesurface, and the device may be used in a lit environment/room to imagewhite light reflectance/fluorescence. The device may also be used in adim or dark environment/room to optimize the tissue fluorescence signalsand to minimize background signals from room lights. The device may beused for direct (e.g., with the unaided eye) or indirect (e.g., via theviewing screen of the digital imaging device) visualization of woundsand surrounding normal tissues.

The device may also be embodied as not being handheld or portable, forexample as being attached to a mounting mechanism (e.g., a tripod orstand) for use as a relatively stationary optical imaging device forwhite light, fluorescence and reflectance imaging of objects, materials,and surfaces (e.g., a body). This may allow the device to be used on adesk or table or for ‘assembly line’ imaging of objects, materials andsurfaces. In some embodiments, the mounting mechanism may be mobile.

Other features of this device may include the capability of digitalimage and video recording, possibly with audio, methods fordocumentation (e.g., with image storage and analysis software), andwired or wireless data transmission for remote telemedicine/E-healthneeds.

In some embodiments, the image acquisition device may be a mobiledevice, such as a cellular telephone or smartphone. In theseembodiments, the mobile device is used to obtain the white light and/orfluorescent images. As discussed further below, the image acquisitiondevice may also include an adaptor for attachment to the mobile device.The insets e) and f) of FIG. 4 show an embodiment where the imageacquisition device is a mobile communication device, such as, forexample, a cellular telephone. The cellular telephone used in thisexample is a Samsung Model A-900, which is equipped with a 1.3 megapixeldigital camera. As illustrated in FIG. 4 , the telephone is fitted intothe holding frame for convenient imaging. Inset e) shows the use of thedevice to image a piece of paper with fluorescent ink showing the word“Wound”. Inset f) shows imaging of fluorescent ink stained fingers, anddetection of the common skin bacteria P. Acnes. The images from thecellular telephone (or smartphone) may be sent wirelessly to anothercellular telephone, (smartphone) or wirelessly (e.g., via Bluetoothconnectivity) to a personal computer for image storage and analysis.This demonstrates the capability of the device to perform real-timehandheld fluorescence imaging and wireless transmission to a remotesite/person as part of a telemedicine/E-health wound careinfrastructure.

In order to demonstrate the capabilities of the image acquisition devicein wound care and other relevant applications, a number of feasibilityexperiments were conducted using the particular example described above.It should be noted that all fluorescence imaging experiments used a Sonycamera (Sony Cybershot DSC-T200 Digital Camera, Sony Corporation, NorthAmerica) as described above. The camera settings were set so that imageswere captured without a flash, and with the ‘Macro’ imaging mode set.Images were captured at 8 megapixels. The flash was used to capturewhite light reflectance images. All images were stored on the xD memorycard for subsequent transfer to a personal computer for long-termstorage and image analysis.

In one exemplary embodiment, white light reflectance and fluorescenceimages/movies captured with the device were imported into AdobePhotoshop for image analysis. However, image analysis software wasdesigned using MatLab™ (Mathworks) to allow a variety of image-basedspectral algorithms (e.g., red-to-green fluorescence ratios, etc.) to beused to extract pertinent image data (e.g., spatial and spectral data)for quantitative detection/diagnostic value. Image post-processing alsoincluded mathematical manipulation of the images.

In accordance with another exemplary embodiment of the presentdisclosure, a handheld device for collection of data from a woundincludes a low-cost, consumer-grade, Super HAD™ charge-coupled device(CCD) sensor-based camera (Model DSC-T900, Sony Corp., Japan), with a 35to 140 mm equivalent 4× zoom lens housed in a plastic body and poweredby rechargeable batteries. An exemplary embodiment of this handheldimaging device is shown in FIG. 5A. Inset (a) of FIG. 5A is a view ofthe user-facing side of the device showing a wound fluorescence (FL)image displayed in real time on a liquid-crystal (LC) display screen 100in high definition. As shown in inset (a) of FIG. 5A, the deviceincludes a housing 110 attached to a handle 120. The housing 110 may beplastic, or any conventional material well-known in the art.Additionally, the handle 120 may be connected to a power cable 130 forpower, as discussed above. The housing 110 may include an image capturebutton 140, to control the image captured on the display screen 100, andan on/off switch 150. As shown in inset (a) of FIG. 5A, the device mayalso include heat dissipating fans 160 to allow excess heat to beremoved from the device, if required. Furthermore, a toggle switch 170may be provided so that a user can switch between white light imagingand fluorescent imaging.

Inset (b) of FIG. 5A is a view of the patient-facing side of the deviceshowing a dual excitation LED array assembly 180 that includes anoptical filter. The LED array assembly 180 may be white light (WL) and405-nm LED arrays that provide illumination of the wound. The WL LEDsmay be broadband LEDs that are electrically powered by a standard AC125V source and that provide illumination during WL imaging. The FL LEDsmay be two monochromatic violet/blue (λ=405 nm+/−20 nm) LED arrays thatprovide 4 Watt excitation light power during FL imaging. WL and FLimages are detected by a high-sensitivity CCD sensor mounted with a dualband FL filter in front of the camera lens to block excitation lightreflected from the skin surface. Additionally, the device may include adual white light LED array 185 coupled to an iris 187. A FL emissionfilter 190 may also be disposed on the back side of the device. The FLemission filter 190 may be placed in front of the CCD sensor.

The device of FIG. 5A is configured to collect high-resolution 12.1Mpixels color WL and AF images (or videos) in real time (<1 s), whichare displayed in red-green-blue (RGB) format on a 3.5-in.touch-sensitive color liquid-crystal display (LCD) screen of the device.The device includes broadband white light-emitting diodes (LEDs),electrically powered by a standard AC125V source, configured to provideillumination during WL imaging. The two arrays 180, 187 may bemonochromatic violet-blue (kexc=405_20 nm) LED arrays (Model LZ4,LedEngin, San Jose, California) to provide 4-W excitation light powerduring FL imaging (bright, uniform illumination area ˜700 cm2 at cmdistance from skin surface). The WL and FL images are detected by ahigh-sensitivity CCD sensor mounted with a dual band FL filter(kemiss=500 to 550 and 590 to 690 nm) (Chroma Technologies Corp.,Vermont) in front of the camera lens to block excitation light reflectedfrom the skin surface. The FL emission filter 190 is configured tospectrally separate tissue and bacteria AF. The device is configured todisplay the spectrally separated tissue and bacterial AF as a compositeRGB image without image processing or color-correction, thus allowingthe user to see the bacteria distribution within the anatomical contextof the wound and body site. The CCD image sensor is sensitive acrossultraviolet (<400 nm), visible (400 to 700 nm), and near-infrared (700to 900 nm) wavelengths to AF of tissues and bacteria, in the absence ofexogenous contrast agents.

FIG. 5B shows another exemplary embodiment of the device in use with anendoscope 42. Endoscope 42 may be flexible or rigid. As shown in FIG.5B, endoscope 42 may be attached to the device to obtain FL and/or whitelight images of anatomically-constrained locations (e.g., hard to reachlocations located in the head and neck), such as within body lumens of apatient. The device, when used with endoscope 42, may include multipleexcitation LED arrays configured, for example, for sequenced 405 nm, 532nm, etc, illumination for multiplexed read out of the microarraybioassay. Endoscope 42 may also provide 3D stereoscopic fluorescenceimaging that may provide, for example, topography-specific informationabout bacterial infection of curved surfaces.

In another exemplary embodiment, the image acquisition device is ahandheld device that is incorporated with a mobile device to take bothwhite light images and fluorescent images. It is also contemplated thatin some embodiments, the handheld device takes only white light imagesor only fluorescent images when incorporated with the mobile device. Themobile device may be a mobile communication device, such as asmartphone, mobile phone, iPod, iPhone, or other such device havingexisting image-capturing capabilities such as the CCD sensor. Althoughdescribed herein with regard to usage with the iPod touch or iPhone, itshould be understood that other platforms (e.g., Android, etc.) may beused. For example, as shown in FIG. 6A, the device incorporates aniPhone 4S. The handheld device may also have one or more downloadableapplications, enabling the user to take the white light and/orfluorescent images. Those of ordinary skill in the art will understandthat the mobile communication devices described and illustrated hereinare exemplary only, and that various other types and/or configurationsof image acquisition devices, handheld devices, and/or mobilecommunication devices are contemplated without departing from the scopeof the present disclosure and claims.

A mobile imaging device adaptor 200 is shown in FIG. 6A. The adaptor 200is a handheld imaging adaptor for a mobile device that providespoint-of-care, real-time wound care assessment and management. Theadaptor 200, when used with the mobile device, is a non-invasive devicethat allows clinicians and nurses to collect white light and/orfluorescence digital images. In some embodiments, the adaptor 200 isconfigured to collect both white light and fluorescent images. Thus, theadaptor 200 may include a toggle switch to switch between the whitelight and fluorescent imaging modes. In other embodiments, the adaptor200 is configured to collect fluorescent images and white light imagesare captured by the mobile device when the adaptor is removed from themobile device.

FIG. 6A shows an embodiment in the which the adaptor 200 is configuredfor fluorescence imaging and is coupled to a mobile device. When in thefluorescence imaging mode, the mobile device and adaptor 200 detect thepresence of clinically relevant bacteria in, for example, a wound bed,wound periphery, and off-site area while also enabling visualization ofconnective tissue to provide important anatomical context to the userwith respect to the location of the bacteria. In this manner, contrastagents are not required when using the mobile device and adaptor.

Inset (a) of FIG. 6A shows a front view of the device, showing theoptical components and battery holder of the accessory adaptor, which ismounted onto a standard iPhone 4S smart phone. As shown in inset (a),the front side of the adaptor 200 includes a dichroic mirror 210, an LEDexcitation filter 220, an emission filter and macro lens 230, and abattery holder 240. Inset (b) of FIG. 6A shows a back view of thedevice, showing the on/off power switch 250 and the LCD display screen260 on the mobile device, on which the WL and FL images are viewed bythe user. Additionally, the adaptor 200 may include an LED heat sink270.

White light imaging allows the user to capture an image of a patientwound, and the fluorescence imaging allows the user to capture acorresponding image highlighting the presence of bacteria on the image.Both white light images and fluorescence images are viewed on thedisplay screen 260 of the mobile device. Thus, the display screen 260may be coupled to a camera on the mobile device. The display screen 260may range between about 4-inches (diagonal) and about 7-inches(diagonal) widescreen display with Multi-Touch IPS technology. Othersize displays may be used based on user needs. In one example, thedisplay quality settings are 1136×640-pixel resolution at 326 pixels perinch; 800:1 contrast ratio; and 500 cd/m2 max brightness. The displaymay have a fingerprint-resistant oleophobic coating. The resolution ofthe camera may be about 5 Megapixels and may have resolutions higherthan 5 Megapixels, such as up to about 24 Megapixels, depending uponavailability, amount of storage available, etc. The selection of thelens design allows the production of high-quality images, specificallyin the red and green spectra. In one exemplary embodiment, afive-element lens is used (as iPod touch design). The user can tap tofocus video and/or still images. The camera has optimal performance inthe dark. The camera has an LED flash and shutter speeds are high.

As shown in FIGS. 6A and 6B, the exemplary embodiment of the handhelddevice integrates a consumer grade mobile phone camera with a customoptical platform. The image acquisition occurs on the mobile phonecamera and functions independently of the device housing, electronicsand optics. The images are displayed on the phone's LCD touch screen andare stored on the phone itself. The customized optical design includesone violet 405 nm LED 270 positioned at a 45-degree angle to thedichroic mirror 210, which is fixed in front of the camera sensor. Thedichroic mirror 210 reflects violet light while transmitting all greaterwavelengths to produce fluorescence excitation illumination that iscoaxial to the camera sensor. A macro lens 280 is situated in front ofthe camera sensor to allow for focused close-up imaging of wounds (<10cm). A specific combination of excitation and emission filters are usedto capture the red and green fluorescence data that is indicative ofbacteria and connective tissues respectively. Emission filters may beused to block excitation light from a camera sensor of the phone. Theadaptor may include a 9V battery to power the violet LED 270, which istriggered by the user through an external power switch. A heat sink 290is attached to the back of the device for the LED printed circuit boardwith thermal paste to effectively transfer and dissipate the heatgenerated by the 5 W violet LED 270.

The adaptor includes a fluorescent light source as discussed above. Thefluorescent light source may be a violet light source that may emitexcitation light in the range of about 400 nm-about 450 nm. It is alsocontemplated that the fluorescent light source emits excitation light inthe range of about 450 nm-about 500 nm, about 500 nm-about 550 nm, about600 nm-about 650 nm, about 650 nm-about 700 nm, about 700 nm-about 750nm, and combinations thereof.

In accordance with this exemplary embodiment, the housing 205 of theadaptor 200 may be made by 3D printing. Other types of suitablestructures are disclosed herein, and variations thereof will beunderstood by those of ordinary skill in the art based on the presentteachings. The housing 205 provides a means for aligning the opticalcomponents with a consumer grade camera and encasing both the electricalcomponents used to drive the LED and the thermal solution while creatinga user friendly and lightweight handheld design. As shown in FIGS. 6Aand 6B, the housing 205 may include an extension portion 206 thatextends outwardly from a patient-facing side of the adaptor. Thedichroic mirror 210, the LED excitation filter 220, and the emissionfilter and macro lens 230 may be disposed within the extension portion206. Furthermore, the extension portion 206 may be aligned with thecamera (optical sensor) of the mobile device so that the fluorescentemissions of the target illuminated by the fluorescent excitation lightare directed to the camera (optical sensor). Further, as shown in FIG.6B, the use of the dichroic mirror 210 allows the fluorescent lightsource positioned below the camera (optical sensor) to be directed outof the extension 206 toward the target being imaged. The filter in frontof the camera blocks the reflected excitation light while allowing otherwavelengths of light to pass through to the camera. For example, theemission filter may be a dual band high transmission bandpass filterthat is configured to pass emissions having wavelengths corresponding tothe dual bands of the filter.

FIG. 6C shows a rear, perspective view of the adaptor 200 without themobile device. As shown in FIG. 6C, the adaptor 200 includes an opening207 so that the adaptor 200 is designed to slide onto the top of themobile device, for example the iPhone 4s, and fit snuggly around themobile device to remain fixed in place during imaging. The adaptor 200is removable from the mobile device. Thus, in some embodiments, theadaptor 200 may be removed from the mobile device for white lightimaging, in which the flash of the camera of the mobile device is usedas the white light source for white light imaging. In accordance withanother exemplary embodiment, the adaptor 200 may be permanently affixedto the mobile device, such as the iPhone 4s. In such an embodiment, amovable filter may be provided for switching between white light imagingand fluorescent imaging, in a manner similar to that described withregard to embodiments of the handheld device discussed in FIGS. 1 and 2. Thus, in this embodiment, a toggle switch may be provided to switchbetween the white light and fluorescent imaging.

As shown in FIG. 6C, the opening 207 of the adaptor 200 may be sized andconfigured to receive a mobile device such as a cellular phone. Theopening may be sized for a specific mobile device, for example for theiPhone 4s. Thus, a top portion of the mobile device, including thecamera on the mobile device, is slid into the opening 207 on the adaptor200 to align the camera of the mobile device with the filters and thedichroic mirror 210 on the adaptor 200, as shown in FIG. 6B. The adaptor200 in the embodiment of FIG. 6C also includes a switch 250 to turnon/off the LED excitation light. The switch 250 may also be used toswitch between a white light imaging and a fluorescent imaging mode, asdiscussed above.

FIG. 6D shows another exemplary embodiment of an adaptor 300 for usewith a mobile device, such as, for example, the iPhone 4s. As shown inFIG. 6D, the adaptor 300 includes an opening 307 that is sized toreceive the entire mobile device. In this embodiment, the back and sideportions of the mobile device are disposed in the opening 307 of theadaptor. An extension portion 306 is positioned over the camera (opticalsensor) of the mobile device in order to align the camera (opticalsensor) of the phone with the filter(s), to allow fluorescenceexcitation light produced by the fluorescent LED to be directed towardan imaging target, and to allow the camera sensor to receive theresultant fluorescence emissions. The embodiment of FIG. 6D alsoincludes a toggle switch 350, to turn on/off the LED excitation light,as discussed above.

To perform fluorescence imaging using the adaptor 200, 300, the userswitches on the violet LED using the toggle switch on the back of thedevice (FIGS. 6A, 6C, 6D). As the switch is moved to the ‘on’ position,the 9V battery sends power to the LED to drive the violet LED. Theviolet broad band LED, which is situated perpendicularly to the iPhonecamera sensor and 45 degrees to the dichroic mirror, emits 405 nm lightat the dichroic mirror. The dichroic mirror reflects almost 100% of thelight at the 405 nm wavelength directly onto the target. The tissues andbacteria in the target absorb the 405 nm photons from the violet LED,and photons of a longer wavelength are then emitted by the bacteria andtissue to create fluorescence. The specific emission filter that ispositioned in front of the mobile device's camera sensor controls thewavelengths of photons that are able to reach the camera sensor andeffectively blocks the excitation light. The mobile device's camerasensor captures an RGB image of the emitted photons where bacteria isdisplayed as red (e.g. S. aureus) or very bright bluish-green (e.g.Pseudomonas aruginosa) and healthy connective tissues from skin orwounds are captured by a green fluorescence signal. The user may thenuse the fluorescence image (or video) stored on the mobile device todetermine where bacteria are located within and around a wound.

Those of ordinary skill in the art will understand that the adaptorsdescribed and illustrated herein are exemplary only, and that variousother types and/or configurations of adaptors are contemplated withoutdeparting from the scope of the present disclosure and claims.

EXAMPLES

The studies discussed herein aimed to determine the ability of thehandheld device to accurately detect and measure bacterial wounds inreal time, guide treatment decisions, and track wound healing over thecourse of antibacterial treatment.

In one example, a study using the handheld device described hereintracked patient wounds over time. The study was broken into two parts,the first part to establish the safety and feasibility of AF imaging toimprove wound sampling by accurately detecting clinically-significantlevels of pathogenic bacteria in chronic wound patients, compared tostandard wound assessment (including swab-based methods). The secondpart to demonstrate the feasibility of AF image guidance for woundtreatment and quantitative treatment response, compared to standardwound assessment (including swab-based methods). Swab cultures were usedto compare AF imagining with WL examination, to determine sensitivity,specificity, and predictive values for FL imaging for detectingclinically-significant bacterial loads.

In the first part of the study, high resolution WL and FL images weretaken of every patient's wound at each visit. A disposable lengthcalibration scale (sticker) was placed near the wound during WL and FLimaging to track each patient's ID and date of the imaging. Regular roomlighting was used during WL imaging, and the lights were turned offduring FL imaging to eliminate any artifacts in the images. To preservebacterial characteristics on the tissue, no swabs were taken of thewound until completion of both the WL and FL imaging. The process tocapture a WL image took 1-2 min per wound, and subsequent FL imagingtook 1-2 min per wound. The clinician also swabbed each suspiciousmarked area on the patient using the Levine sampling method, and swabswere sent for blinded microbiology testing (it is noted that the Levinesampling method is the most commonly used swabbing method and involvesonly sampling the center of the wound). Patients were treated anddischarged according to standard protocols.

The location(s) of red and/or green AF were marked on printed images, asdiscussed further below. FL spectroscopy was used in some cases tocharacterize AF areas in/around the wound. Spectra were compared on alocation basis with microbiology results. A complete data file for eachpatient's visit (CSS, WL and FL images, spectroscopy and microbiology)were stored in an electronic database according to Good ClinicalPractice guidelines.

In the second part of the study, three sequential 2-month arms wereused: non-guided treatment (control), FL guided treatment and non-guidedtreatment (control). In the first 2-month phase, wounds were assessedweekly by CSS and then treated at the discretion of the clinical teamusing best practice methods (ultrasonic and/or scalpel wounddebridement, topical/systemic antibiotics, saline wash, dry oranti-microbial dressings or iodine). Corresponding WL and FL images weretaken of each wound pre- and post-treatment as described previously.2-month evaluation periods were selected based on established clinicaldata for venous leg ulcers showing that this is sufficient to detect areliable and meaningful change in wound area, as a predictive indicatorof healing. Wound swabs were collected by FL guidance. Clinicians wereblinded to FL images during this first (control) phase. During thesubsequent 2-month phase, wound assessment was performed normally butclinicians were shown FL images of the wound during treatment.

During the final 2-month phase, WL and FL imaging were performed andswabs were collected, with clinician blinding to the FL results duringtreatment delivery. Importantly, while the clinicians understood andcould remember the meaning and characteristics of the red and greenfluorescence signals, respectively, blinding them during treatmentdelivery in the control periods was possible because the fluorescenceresults for each wound examination and each patient were different.Thus, in the absence of real-time fluorescence guidance during woundtreatment, previous knowledge of fluorescence characteristics did notsubstantively influence the treatment decisions during the controlperiods. WL and FL images were also taken after each treatment toanalyze wound area.

Four blinded, trained clinical and/or research staff membersindependently measured the average wound size on WL images using digitaltracing (MATLAB v.7.9.0, The MathWorks, Massachusetts, USA). Theobservers measured the wounds in separate sessions with a minimum of 7days between sessions to minimize memory effect. An adhesive scale barplaced adjacent to the wound during imaging provided accurate lengthcalibration within +0.5 mm. Room lights remained on during WL imaging,but were turned off during FL imaging. WL and FL images were collectedwith the handheld device held/positioned 10-15 cm from the wound. Allimaging parameters (distance, exposure time, ISO setting, white balance,etc.) were kept constant between visits. For distances less than 5 cmfrom a wound (small diameter wounds), the camera's built-in macro modewas used. Automatic focusing allowed rapid (˜1 s) image acquisition.Images (or video) were captured in real-time and stored on the camera'smemory card. Switching between WL and FL modes was substantiallyinstantaneous using a built in “toggle switch.” Devices weredecontaminated between uses with 70% ethyl alcohol.

WL and AF images were transferred to a laptop. Regions of interest(ROIs) were identified from individual 1024×1024 pixel FL images of eachwound at each clinic visit. RGB images were separated into individualchannels. The green and red channels of the RGB image wererepresentative of the true tissue and bacterial AF signals detected invivo. To quantify bacterial levels from individual FL images, thefollowing image processing procedures were used. Briefly, individualgreen and red image channels from each RGB image were converted togreyscale (the blue channel was not used) and pixels whose greyscaleintensity was above a given histogram threshold (selected to reduce thebackground noise of the raw image) were counted. A red color mask forred FL bacteria was created by finding the local maxima in the colorrange 100-255 greyscale. Then, an inverted green color mask was used toremove the green FL. All pixels with red FL (above the histogramthreshold) were binarized and the sum of all “1” pixels was calculated.This was repeated for the green channel of each image. These data gavean estimate of the amount of red (or green) bacteria in each image. Thenumber of FL pixels was converted into a more useful pixel area measure(cm2) using the adhesive length calibration stickers, thereby providingthe total amount of fluorescent bacteria as an area measurement. TheLevine method was used to aseptically swab wounds for confirmation ofbacterial presence, species typing, Gram signing, antibiograms, andsemi-quantitative bacterial load.

Tissue AF produced by endogenous collagen or elastin in the skinappeared as green FL, and clinically-relevant bacterial colonies (e.g.Staphylococcus aureus) appeared as red FL (caused by endogenousporphyrins). Some bacteria (e.g. Pseudomonus aeruginosa) produced ablue-green signal, due to siderophores/pyoverdins, which wasdifferentiated spectrally and texturally from dermis AF using imageanalysis software. WL and FL images were collected in less than 1 secondby the high-sensitivity CCD sensor mounted with a dual band FL filter(Xemiss=500-550 and 590-690 nm) (Chroma Technologies Corp, VT, USA). TheCCD image sensor was sensitive across a broad wavelength range of˜300-800 nm. The handheld device integrated easily into the routineclinical work flow. By combining tissue FL with bacterial FL in a singlecomposite image, the clinician instantly visualized the distribution andextent of the bacterial load within the anatomical context of the woundand body site. Typically, FL imaging added approximately 1-3minutes/patient to the wound assessment routine, depending on the numberof wounds and the duration of FL-guided swabbing.

The variation in measurements of wound areas between images taken underWL and FL were compared. The correlation between change in average woundarea and FL image-guided treatment using Pearson correlationcoefficients was also calculated. Assessing changes in wound areabetween the first control, the second FL image-guided, and the thirdcontrol periods were performed using a linear mixed effect model.

The accuracy of identifying clinically-significant bacterial load for AFimage-guided was compared with swabbing techniques and WL imaging. Atotal of 490 swabs were collected, of which 36.9% were taken from woundbeds, 30.2% from wound peripheries, and 32.9% from “off-site” areas. Itwas determined that the AF accurately determined 74.5% of wounds withclinically-significant bioburden, and that WL imaging only detected52.5% of the wounds. The handheld device accurately determinedclinically-significant bioburden 82.4% of the time in the woundperiphery and 67.1% in other areas. WL examination was correct only17.6% of the time in peripheries and 32.9% in other areas. The overallaccuracy of judging the presence of clinically-significant bacterial inchronic wounds for AF was 74.5% versus 35.5% for traditional methods ofWL and swab results.

AF imaging detected clinically significant bacterial load in 85% ofwound peripheries missed by conventional methods. Thus, the Levinemethod for swabbing only the wound bed may be insufficient, possiblyresulting in antibacterial treatment being inappropriately withheld.However, modifying standard sampling practices to include swabbing ofthe wound periphery of all wounds would be impractical and costly. AFimaging could help clinicians decide if and where wound margins requiresampling. The handheld imaging device also identified clinicallysignificant bioburden in surrounding locations close to wounds, whichrepresent sites of potential re-infection, where traditional methods donot examine or swab.

Identifying and quantitating wound bacterial burden is an importantdeterminant of infection and healing. Data on the visualization andquantitative tracking of bacterial load led to the identification of anew, simple method for image-guided debridement and topical applicationof antibiotic and antiseptic, which minimizes unnecessary trauma to thewound boundary and maximizes the contribution of debridement to reducingbacterial burden. Every wound has the potential for infection, butdistinguishing true infection from critical colonization by bestpractice methods remains challenging and arbitrary, and can lead toover- and under-treatment.

The handheld imaging device identifies pathogenic microbes anddifferentiates between at least the two major pathogenic species (P.aeruginosa and S. aureus), and informs medical treatment decisions. Thedevice also offers a quantitative and reproducible way to monitor theeffectiveness of existing and emerging wound care treatments.Furthermore, the handheld imaging device can be used to diagnosecritically colonized wounds.

Multiple variables including host response, local and systemic factors,malperfusion, immunosuppression, diabetes, and medications affect therisk of infection. Critically colonized wounds can be difficult todiagnose because they do not always display classical signs of infectionor clearly elevated levels of bioburden. Indeed, the clinical relevanceof differentiating critically colonized wounds from infected woundsremains controversial. Identifying a high bacterial load in asymptomaticpatients before infection occurs using AF imaging may help preventinfections by prompting aggressive treatment. If a bacterial infectionis suspected, antibiotic selection could be guided by the establishedclinical principles and by AF identification of heavy bacterial burdenand differentiation between Gram negative P. aeruginosa and Grampositive S. aureus.

In another exemplary embodiment, image analysis may be carried out onthe handheld device or WL and FL images may be transferred to a laptopfor image processing. Image analysis and processing of image data may beperformed using a processor of the handheld device, and the results ofsuch analyses may be displayed on the display of the handheld device.

The following two exemplary programs may be used for image processing(for example, analysis of the data collected by the exemplary deviceusing the Super HAD™ charge-coupled device (CCD) sensor-based camera(Model DSC-T900)) and portions of these processes are illustrated inFIGS. 7A and 7B: MATLAB software (Version 7.9.0, The MathWorks,Massachusetts) using a custom-written program and ImageJ Software(Version 1.45n). In the MATLAB program, regions of interests (ROIs) areidentified from individual 1024×1024 pixel FL images of each wound. RGBimages are separated into individual channels. Green (500 to 550 nmemission) and red AF (>590 nm) from tissue components and bacteria,respectively, detected by the CCD sensor are naturally alignedspectrally with the red and green filters on the Sony CCD image sensor.Thus, the green and red channels of the RGB image displayed on thehandheld device's LCD screen are representative of the true tissue andbacterial AF signals detected in vivo. To quantify bacterial levels fromindividual FL images, the following image processing procedures may beused. Briefly, individual green and red image channels from each RGBimage are converted to gray scale (the blue channel is not used) andpixels whose gray scale intensity is above a given histogram threshold(selected to reduce the background noise of the raw image) are counted.In certain embodiments, it is possible the blue channel would be used,for example, when imaging the amount of 405 nm excitation light that isabsorbed by tissues/blood when imaging tissue vascularity/perfusion.

A red color mask for red FL bacteria is created by finding the localmaxima in the color range 100 to 255 gray scale. Then, an inverted greencolor mask is used to remove the green FL. All pixels with red FL (abovethe histogram threshold) are binarized and the sum of all “1” pixels iscalculated. This is repeated for the green channel of each image. Thesedata give an estimate of the amount of red bacteria in each image. Thenumber of FL pixels is converted into a more useful pixel area measure(cm2) by applying a ruler on the pixel image, thereby providing thetotal amount of fluorescent bacteria as an area measurement (cm2). Thesizes of the wounds may be traced and measured similarly by convertingpixel areas to cm2 of the circled wound area on the WL images. Theresolution of the FL images is sufficient to localize bacteria based onregions of FL. ImageJ software may be used to separate FL images intored, green, and blue channels using the built-in batch processingfunction “Split Channels” located within the image menu and colorsubmenu of the camera. Each resulting channel is displayed and saved ingray scale. For further analysis, an ROI may be identified in eachcorresponding red, green, and blue channel image. Under the built-inanalysis menu, the “Set Measurement” function may be used to select andmeasure the following measurement parameters for each color channelimage: pixel area, min. and max. gray scale intensity values, and meangray intensity values. The average red channel intensity value may bedetermined as (bacterial) FL intensity per square pixel in each redchannel image and then used for data analysis and comparison.

In one exemplary embodiment, a mouse skin wound model was used tocorrelate wound status with the progression of bacterial infection (n=5;8 to 12 weeks; NCRNU-F). Correlation was based on data obtained usingthe exemplary handheld device described above, which incorporates theSuper HAD™ charge-coupled device (CCD) sensor-based camera (ModelDSC-T900. Daily WL and FL images were taken of the wounds as they becameinfected over time. Antibacterial treatment (topical Mupirocin threetimes daily, for a total of 1 day) was applied to the wound site whenthe red FL intensity peaked. The anti-microbial effect of the treatmentwas monitored over time using the handheld device to acquire daily WLand FL images of the wound after treatment. The wounds were monitoredfor a total of 10 days (see FIG. 8 ), after which the mice weresacrificed. Bacterial amounts from FL images and wound size from WLimages were quantified using the MATLAB program described above andcompared over time to determine the wound healing status.

FIG. 8 shows representative WL and FL images for a single mouse trackedover 10 days. Inset (a) of FIG. 8 provides images taken with a handhelddevice in accordance with the present disclosure and showing the twoequal-sized wounds on both sides of the spine. The right wound wasinoculated with S. aureus in PBS and the left wound was inoculated withPBS only (control). The top row shows WL images, the middle row shows FLimages, and the bottom row shows MATLAB quantified images, correspondingto bacterial areas and intensities. The FL imaging data demonstrated asignificant increase in bacterial FL intensity in the wound inoculatedwith S. aureus, compared with the control wound, peaking on day 6.Mupirocin (day 7, red arrow) significantly decreased bacterial FL on day8 to almost zero, indicating treatment effect. Bacteria increased againon days 9 and 10. Inset (b) of FIG. 8 provides a graph showingquantitative changes in bacterial load from FL images obtained in inset(a) of FIG. 8 .

In accordance with another exemplary embodiment, BLI can be used tomeasure the absolute amount of bacteria in vivo, because it is one ofthe most sensitive and reliable screening tools for determiningbacterial load. BLI collects the light emitted from the enzymaticreaction of luciferase and luciferin and therefore does not requireexcitation light. FL imaging using the handheld device (without anyexogenous FL contrast agent administration) and BLI imaging ofinoculated S. aureus bacteria were tracked over time and the FL and BLIintensities were compared (see FIG. 9 ) (n=7). The bacterial BLI signaldid not contribute to the FL signal detected by the handheld device'sconsumer grade-CCD camera. Gram-positive bioluminescent S. aureus-Xen8.1from the parental strain S. aureus 8325-4 (Caliper) was grown tomid-exponential phase the day before pathogen inoculation. Bacteria withthe BLI cassette produce the luciferase enzyme and its substrate(luciferin), thereby emitting a 440 to 490 nm bioluminescent signal whenmetabolically active (FIG. 9 ). The bacteria (1010 CFU) were suspendedin 0.5 mL of PBS and injected into the wounds of female athymic nudemice (n=7; 8 to 12 weeks; NCRNU-F Homozygous). To detect S. aureusbioluminescence, BLI images of the wound were acquired before,immediately after, and 1, 2, 3, 4, 5, 6, and 7 days postinoculationinside the dark chamber of the Xenogen IVIS Spectrum Imaging System 100(Caliper, Massachusetts), using an exposure time of 10 s. BLI imageswere captured using Living Image In Vivo Imaging software (Caliper,Massachusetts). ROIs were digitally circumscribed over the wound and thetotal luminescence intensity counts were measured within the ROIs foreach time point imaged. The absolute amount of bacteria measured fromthe BLI signals was tested for correlation with the corresponding FLsignals on the FL images taken over time of the same wound using thehandheld device (as described above).

FIG. 9 provides preclinical data which show that pathogenic bacterialautofluorescence (AF) intensity correlates with bacterial load in vivo.Inset (a) of FIG. 9 shows a time course prototype device mobile imagesof skin wounds in a mouse prior to and after inoculation withbioluminescent S. aureus-Xen8.1 (10¹⁰ CFU in 30 μL PBS). RepresentativeWL (top row), AF (middle row), and bioluminescence (bottom row) imagesare shown for each time point to 7 days after inoculation in a woundedmouse. BLI imaging gives absolute bacterial amount in vivo. Red arrowsshow when the tegaderm bandage was exchanged, causing some bacteria tobe removed from the surface. Inset (b) of FIG. 9 shows average red FLfrom S. aureus-Xen8.1 (n=7 nude mice) shown as a function of timedemonstrating an increase in daily S. aureus bacterial FL (calculatedfrom red channel of RGB images using ImageJ software). At days 2 and 7,tegaderm bandages were exchanged as per animal protocol. Averagebacterial FL peaked at day 4 postinoculation. Inset (c) of FIG. 9illustrates a corresponding time course bioluminescence data (calculatedfrom ROI) show similar increase and peaking at day 4 in total bacterialload in the wound. Data indicates a strong positive correlation (Pearsoncorrelation coefficient r=0.6889) between total bacterial AF in a woundand the bacterial load in vivo. Standard errors are shown. Scale bars:(a) WL 1.5 cm and AF, BLI 1 cm.

Imaging of Bacteriological Samples

Imaging devices in accordance with the present disclosure may be usefulfor imaging and/or monitoring in clinical microbiology laboratories.Such devices may be used for quantitative imaging of bacterial coloniesand quantifying colony growth in common microbiology assays.Fluorescence imaging of bacterial colonies may be used to determinegrowth kinetics. Software may be used to provide automatic counting ofbacterial colonies.

To demonstration the utility of such devices in a bacteriology/culturelaboratory, live bacterial cultures were grown on sheep's blood agarplates. Bacterial species included Streptococcus pyogenes, Serratiamarcescens, Staphylococcus aureus, Staphylococcus epidermidis,Escherichia coli, and Pseudomonas aeruginosa (American Type CultureCollection, ATCC). These were grown and maintained under standardincubation conditions at 37° C. and used for experimentation when during‘exponential growth phase’. Once colonies were detected in the plates(˜24 h after inoculation), the device was used to image agar platescontaining individual bacterial species in a darkened room. Usingviolet/blue (about 405 nm) excitation light, the device was used toimage both combined green and red autofluorescence (about 490-550 nm andabout 610-640 nm emission) and only red autofluorescence (about 635+/−10nm, the peak emission wavelength for fluorescent endogenous porphyrins)of each agar plate. Fluorescence images were taken of each bacterialspecies over time for comparison and to monitor colony growth.

Reference is now made to FIG. 10 . Inset a) of FIG. 10 shows a devicebeing used to image live bacterial cultures growing on sheep's bloodagar plates to detect bacterial autofluorescence. Inset b) of FIG. 10shows the image of autofluorescence emitted by Pseudomonas aruginosa.The device may also be used to detect, quantify and/or monitor bacterialcolony growth over time using fluorescence, as demonstrated in inset c)of FIG. 10 with fluorescence imaging of the growth of autofluorescentStaphylococcus aureus on an agar plate 24 hours after innoculation. Notethe presence of distinct single bacterial colonies in the lower image.Using violet/blue (e.g., 405 nm) excitation light, the device was usedto detect both combined green and red (e.g., 490-550 nm+610-640 nm) andonly red (e.g., 635+/−10 nm, the peak emission wavelength forfluorescent endogenous porphyrins) emission autofluorescence fromseveral live bacterial species including Streptococcus pyogenes, shownin inset d) of FIG. 10 ; Serratia marcescens, shown in inset e) of FIG.10 ; Staphylococcus aureus, shown in inset f) of FIG. 10 ;Staphylococcus epidermidis, shown in inset g) of FIG. 10 ; Escherichiacoli, shown in inset h) of FIG. 10 ; and Pseudomonas aeruginosa, shownin inset i) of FIG. 10 . Note that the autofluorescence images obtainedby the device of the bacterial colonies may provide useful imagecontrast for simple longitudinal quantitative measurements of bacterialcolonization and growth kinetics, as well as a means of potentiallymonitoring response to therapeutic intervention, with antibiotics,photodynamic therapy (PDT), low level light therapy, hyperbaric oxygentherapy (HOT), or advanced wound care products, as examples.

High spatial resolution of the camera detector combined with significantbacterial autofluorescence signal-to-noise imaging with the deviceallowed detection of very small (e.g., <1 mm diameter) colonies. Thedevice provided a portable and sensitive means of imaging individualbacterial colonies growing in standard agar plates. This provided ameans to quantify and monitor bacterial colony growth kinetics, as seenin inset c), as well as potentially monitoring response to therapeuticintervention, with antibiotics or photodynamic therapy (PDT) asexamples, over time using fluorescence. Therefore, devices in accordancewith the present disclosure may serve as a useful tool in themicrobiology laboratory.

FIG. 11 , inset a), shows an example of the use of an imaging device instandard bacteriology laboratory practice. In inset b) of FIG. 11 ,fluorescence imaging of a Petri dish containing Staphylococcus aureuscombined with custom proprietary image analysis software allowsbacterial colonies to be counted rapidly, and here the fluorescenceimage of the culture dish shows ˜182 (+/−3) colonies (brightbluish-green spots) growing on agar at 37° C. (about 405 nm excitation,about 500-550 nm emission (green), about >600 nm emission (red)).

In addition to providing detecting of bacterial species, the device maybe used for differentiating the presence and/or location of differentbacterial species (e.g., Staphylococcus aureus or Pseudomonasaeruginosa), for example in wounds and surrounding tissues. This may bebased on the different autofluorescence emission signatures of differentbacterial species, including those within the 490-550 nm and 610-640 nmemission wavelength bands when excited by violet/blue light, such aslight around 405 nm. Other combinations of wavelengths may be used todistinguish between other species on the images. This information may beused to select appropriate treatment, such as choice of antibiotic.

Such imaging of bacteriology samples may be applicable to monitoring ofwound care.

Use in Monitoring of Wound Healing

Devices in accordance with the present disclosure may also be scannedabove any wound (e.g., on the body surface) such that the excitationlight may illuminate the wound area. The wound may then be inspectedusing the device such that the operator may view the wound in real-time,for example, via a viewer on the imaging device or via an externaldisplay device (e.g., heads-up display, a television display, a computermonitor, LCD projector or a head-mounted display). It may also bepossible to transmit the images obtained from the device in real-time(e.g., via wireless communication) to a remote viewing site, for examplefor telemedicine purposes, or send the images directly to a printer or acomputer memory storage. Imaging may be performed within the routineclinical assessment of patient with a wound.

Prior to imaging, fiduciary markers (e.g., using an indeliblefluorescent ink pen) may be placed on the surface of the skin near thewound edges or perimeter. For example, four spots, each of a differentfluorescent ink color from separate indelible fluorescent ink pens,which may be provided as a kit to the clinical operator, may be placednear the wound margin or boundary on the normal skin surface. Thesecolors may be imaged by the device using the excitation light and amultispectral band filter that matches the emission wavelength of thefour ink spots. Image analysis may then be performed, by co-registeringthe fiduciary markers for inter-image alignment. Thus, the user may nothave to align the imaging device between different imaging sessions.This technique may facilitate longitudinal (i.e., over time) imaging ofwounds, and the clinical operator may therefore be able to image a woundover time without the need for aligning the imaging device during everyimage acquisition.

In addition, to aid in intensity calibration of the fluorescence images,a disposable simple fluorescent standard ‘strip’ may be placed into thefield of view during wound imaging (e.g., by using a mild adhesive thatsticks the strip to the skin temporarily). The strip may be impregnatedwith one or several different fluorescent dyes of varying concentrationswhich may produce pre-determined and calibrated fluorescence intensitieswhen illuminated by the excitation light source, which may have single(e.g., 405 nm) or multiple fluorescence emission wavelengths orwavelength bands for image intensity calibration. The disposable stripmay also have the four spots as described above (e.g., each of differentdiameters or sizes and each of a different fluorescent ink color with aunique black dot placed next to it) from separate indelible fluorescentink pens. With the strip placed near the wound margin or boundary on thenormal skin surface, the device may be used to take white light andfluorescence images. The strip may offer a convenient way to takemultiple images over time of a given wound and then align the imagesusing image analysis. Also, the fluorescent ‘intensity calibration’strip may also contain an added linear measuring apparatus, such as aruler of fixed length to aid in spatial distance measurements of thewounds. Such a strip may be an example of a calibration target which maybe used with the device to aid in calibration or measuring of imageparameters (e.g., wound size, fluorescence intensity, etc.), and othersimilar calibration target may be used.

It may be desirable to increase the consistency of imaging results andto reproduce the distance between the device and the wound surface,since tissue fluorescence intensity may vary slightly if the distancechanges during multiple imaging sessions. Therefore, in an embodiment,the device may have two light sources, such as low power laser beams,which may be used to triangulate individual beams onto the surface ofthe skin in order to determine a fixed or variable distance between thedevice and the wound surface. This may be done using a simply geometricarrangement between the laser light sources, and this may permit theclinical operator to easily visualize the laser targeting spots on theskin surface and adjust the distance of the device from the wound duringmultiple imaging sessions. Other methods of maintaining a constantdistance may include the use of ultrasound, or the use of a physicalmeasure, such as a ruler, or a range finder mechanism.

Use in White Light Imaging

Devices in accordance with the present disclosure may also be used totake white light images of the total wound with surrounding normaltissues using a measuring apparatus (e.g., a ruler) placed within theimaging field of view. This may allow visual assessment of the wound andcalculation/determination of quantitative parameters such as the woundarea, circumference, diameter, and topographic profile. Wound healingmay be assessed by planimetric measurements of the wound area atmultiple time points (e.g., at clinical visits) until wound healing. Thetime course of wound healing may be compared to the expected healingtime calculated by the multiple time point measurements of wound radiusreduction using the equation R=IA/it (R, radius; A, planimetric woundarea; π, constant 3.14). This quantitative information about the woundmay be used to track and monitor changes in the wound appearance overtime, in order to evaluate and determine the degree of wound healingcaused by natural means or by any therapeutic intervention. This datamay be stored electronically in the health record of the patient forfuture reference. White light imaging may be performed during theinitial clinical assessment of the patient by the operator.

Use in Autofluorescence Imaging

Devices in accordance with the present disclosure may also be designedto detect all or a majority of tissue autofluorescence (AF). Forexample, using a multi-spectral band filter, the device may image tissueautofluorescence emanating from the following tissue biomolecules, aswell as blood-associated optical absorption, for example under 405 nmexcitation: collagen (Types I, II, III, IV, V and others) which appeargreen, elastin which appears greenish-yellow-orange, reducednicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide(FAD), which emit a blue-green autofluorescence signal, andbacteria/microorganisms, most of which appear to have a broad (e.g.,green and red) autofluorescence emission.

Image analysis may include calculating a ratio of red-to-green AF in theimage. Intensity calculations may be obtained from regions of interestwithin the wound images. Pseudo-coloured images may be mapped onto thewhite light images of the wound.

Examples in Wound Healing

Reference is now made to FIG. 12 . A handheld device in accordance withthe present disclosure was tested in a model of wounds contaminated withbacteria. For this, pig meat, with skin, was purchased from a butcher.To simulate wounds, a scalpel was used to make incisions, ranging insize from 1.5 cm² to 4 cm² in the skin, and deep enough to see themuscle layer. The device was used to image some meat samples without(exogenous) addition of bacteria to the simulated wounds. For this, themeat sample was left at room temperature for 24 h in order for bacteriaon the meat to grow, and then imaging was performed with the deviceusing both white light reflectance and autofluorescence, for comparison.

To test the ability of the device to detect connective tissues andseveral common bacteria present in typical wounds, a sample of pig meatwith simulated wounds was prepared by applying six bacterial species toeach of six small 1.5 cm² wound incision sites on the skin surface:Streptococcus pyogenes, Serratia marcescens, Staphylococcus aureus,Staphylococcus epidermidis, Escherichia coli, and Pseudomonasaeruginosa. An additional small incision was made in the meat skin,where no bacteria were added, to serve as a control. However, it wasexpected that bacteria from the other six incisions sites would perhapscontaminate this site in time. The device was used to image thebacteria-laden meat sample using white light reflectance and violet/bluelight-induced tissue autofluorescence emission, using both a dualemission band (450-505 nm and 590-650 nm) emission filter and a singleband (635+/−10 nm) emission filter, on the left and a single band filterover the course of three days, at 24 h time intervals, during which themeat sample was maintained at 37° C. Imaging was also performed on thestyrofoam container on which the meat sample was stored during the threedays.

FIG. 12 shows the results of the device being used for non-invasiveautofluorescence detection of bacteria in a simulated animal woundmodel. Under standard white light imaging, bacteria were occult withinthe wound site, as shown in inset a) of FIG. 12 and magnified in insetb) of FIG. 12 . However, under violet/blue excitation light, the devicewas capable of allowing identification of the presence of bacteriawithin the wound site based on the dramatic increase in red fluorescencefrom bacterial porphyrins against a bright green fluorescence backgroundfrom connective tissue (e.g., collagen and elastins) as seen in inset c)of FIG. 12 and magnified in inset d) of FIG. 12 . Comparison of inset b)and inset d) shows a dramatic increase in red fluorescence frombacterial porphyrins against a bright green fluorescence background fromconnective tissue (e.g., collagen and elastins). It was noted that withautofluorescence, bacterial colonies were also detected on the skinsurface based on their green fluorescence emission causing individualcolonies to appear as punctuate green spots on the skin. These were notseen under white light examination. Fluorescence imaging of connectivetissues aided in determining the wound margins as seen in inset e) andinset f) of FIG. 12 , and some areas of the skin (marked ‘*’ in insetc)) appeared more red fluorescent than other areas, potentiallyindicating subcutaneous infection of porphyrin-producing bacteria.Insets e) and f) also show the device detecting red fluorescent bacteriawithin the surgical wound, which are occult under white light imaging.

The device mapped biodistribution of bacteria within the wound site andon the surrounding skin and thus may aid in targeting specific tissueareas requiring swabbing or biopsy for microbiological testing.Furthermore, using the imaging device may permit the monitoring of theresponse of the bacterially-infected tissues to a variety of medicaltreatments, including the use of antibiotics and other therapies, suchas antibiotics, wound debridement, wound cleaning, photodynamic therapy(PDT), hyperbaric oxygen therapy (HOT), low level light therapy, oranti-matrix metalloproteinase (MMP). The device may be useful forvisualization of bacterial biodistribution at the surface as well aswithin the tissue depth of the wound, and also for surrounding normaltissues. The device may thus be useful for indicating the spatialdistribution of an infection.

ADDITIONAL EXAMPLES

Reference is now made to FIG. 13 . As an example, imaging devices inaccordance with the present disclosure and as discussed above may beused clinically to determine the healing status of a chronic wound andthe success of wound debridement. For example, a typical foot ulcer in aperson with diabetes is shown in the figure, with: (i) the nonhealingedge (i.e., callus) containing ulcerogenic cells with molecular markersindicative of healing impairment and (ii) phenotypically normal butphysiologically impaired cells, which can be stimulated to heal. Despitea wound's appearance after debridement, it may not be healing and mayneed to be evaluated for the presence of specific molecular markers ofinhibition and/or hyperkeratotic tissue (e.g., c-myc and β-catenin).Using the imaging device of the present disclosure in combination withexogenous fluorescently labeled molecular probes against such moleculartargets, the clinician may be able to determine the in-situ expressionof molecular biomarkers. With the device of the present disclosure, oncea wound is debrided, fluorescence imaging of the wound area and imageanalyses may allow biopsy targeting for subsequent immunohistochemistryand this may determine whether the extent of debridement was sufficient.If the extent of debridement was insufficient, as shown in the lowerleft diagram, cells positive for c-myc (which appears green) and nuclearβ-catenin (which appears purple) may be found based on theirfluorescence, indicating the presence of ulcerogenic cells, which mayprevent the wound from healing properly and indicate that additionaldebridement is necessary. Lack of healing may also be demarcated by athicker epidermis, thicker cornified layer, and presence of nuclei inthe cornified layer. If the debridement was successful, as in the lowerright lower diagram, no staining for c-myc or β-catenin may be found,indicating an absence of ulcerogenic cells and successful debridement.These markers of inhibition may be useful, but the goal is actualhealing as defined by the appearance of new epithelium, decreased areaof the wound, and no drainage. This information may be collected usingthe fluorescence imaging device and stored electronically in thepatient's medical record, which may provide an objective analysiscoupled with pathology and microbiology reports. By comparing expectedhealing time with actual healing (i.e., healing progress) time using theimaging device, adaptive treatment strategies may be implemented on aper-patient basis.

FIG. 14 shows an example of the use of a device in accordance with thepresent disclosure for imaging wound healing of a pressure ulcer. Inseta) of FIG. 14 shows a white light image taken with the device of thepresent disclosure of the right foot of a diabetic patient with apressure ulcer. Inset b) of FIG. 14 shows the corresponding fluorescenceimage, which shows the bright red fluorescence of bacteria (bacteriologyresults confirmed presence of heavy growth of Staphylococcus aureus)which are invisible under standard white light examination (yellowarrows). Note the heavy growth of Staphylococcus aureus bacteria aroundthe periphery of the non-healing wound (long yellow arrow). Insets c-d)of FIG. 14 show the spectrally-separated (unmixed) red-green-blue imagesof the raw fluorescence image in inset b), which are used to producespectrally-encoded image maps of the green (e.g. collagen) and red (e.g.bacteria) fluorescence intensities calculated using mathematicalalgorithms and displayed in false color with color scale. Insets f-g) ofFIG. 14 show examples of image-processing methods used to enhance thecontrast of the endogenous bacterial autofluorescence signal bycalculating the red/green fluorescence intensity ratio to reveal thepresence and biodistribution of bacteria (red-orange-yellow) within andaround the open wound. This data illustrates the ability to use customor commercially-available image-analysis software to mathematicallyanalyze the fluorescence images obtained by devices in accordance withthe present disclosure and display them in a meaningful way for clinicaluse, and this may be done in real-time. (Scale bar 1 cm).

FIG. 15 shows an example of the use of a device in accordance with thepresent disclosure for imaging a chronic non-healing wound. Inset a) ofFIG. 15 shows a white light image taken with the device of the presentdisclosure of the left breast of a female patient with Pyodermagangrenosum, shows a chronic non-healing wound (blue arrow) and a healedwound (red arrow). Bacteria typically cannot be visualized by standardwhite light visualization used in conventional clinical examination ofthe wounds. Inset b) of FIG. 15 shows the corresponding fluorescenceimage of the same wounds (in this example, using 405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). Note that thenon-healed wound appears dark colored under fluorescence (mainly due toblood absorption of the excitation and fluorescence emission light),while bacteria appear as punctuate bright red spots in the healed wound(red arrow). Under fluorescence, normal surrounding skin appearscyan-green due to endogenous collagen fluorescence (405 nm excitation).By contrast, the non-healed wound (blue arrow) appears to have a band ofvery bright red fluorescence around the wound border, confirmed withswab cultures (bacteriology) to contain a heavy growth of Staphylococcusaureus (with few Gram positive bacilli and rare Gram positive cocci,confirmed by microscopy). Inset c) of FIG. 15 shows a white light imageof the healed wound in insets a) and b) and inset d) of FIG. 15 showsthe corresponding fluorescence image showing bright red fluorescencefrom bacteria (pink arrows), which are occult under white light. Insete) of FIG. 15 shows a white light image and inset f) of FIG. 15 shows acorresponding fluorescence image of the non-healed breast wound. Notethat bacteria (Staphylococcus aureus) appear to be mainly localizedaround the edge/boundary of the wound (yellow arrow), while lessbacteria are located within the wound (X), determined by thebiodistribution of bacteria directly visualized using fluorescenceimaging, but invisible under white light (black arrow, e). (Scale bar incm).

FIG. 16 further illustrates imaging of a chronic non-healing wound usingan exemplary imaging device in accordance with the present disclosure.Inset a) of FIG. 16 shows a white light image taken with the device ofthe present disclosure of a left breast of a female patient withPyoderma gangrenosum, showing chronic non-healing wound (blue arrow) andhealed wound (blue arrow). Bacteria cannot be visualized by standardwhite light visualization used in clinical examination of the wounds.Inset b) of FIG. 16 shows a corresponding fluorescence image of the samewounds (405 nm excitation, 500-550 nm emission (green), >600 nm emission(red)). While the nipple appears to be normal under white withoutobvious contamination of bacteria, fluorescence imaging shows thepresence of bacteria emanating from the nipple ducts. Swabs of thenipple showed bacteria were Staphylococcus epidermidis (Occasionalgrowth found on culture). (Scale bar in cm)

FIG. 17 shows a central area and border of a chronic non-healing woundimaged using an imaging device in accordance with the presentdisclosure. Inset a) of FIG. 17 shows a white light image taken with thedevice of the present disclosure of a left breast of a female patientwith Pyoderma gangrenosum, showing the central area and border of achronic non-healing wound. Inset b) of FIG. 17 shows a correspondingfluorescence image of the non-healed breast wound (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). Note that bacteria(Staphylococcus aureus; shown by bacterial swabbing) appear to be mainlylocalized around the edge/boundary of the wound, while less bacteria arelocated within the wound (X), determined by the biodistribution ofbacteria directly visualized using fluorescence imaging, but invisibleunder white light. (Scale bar in cm).

FIG. 18 shows further images of a chronic non-healing wound using animaging device in accordance with the present disclosure. Inset a) ofFIG. 18 shows a white light image taken with the device of the presentdisclosure of a left breast of a female patient with Pyodermagangrenosum, showing chronic non-healing wound. Bacteria cannot bevisualized by standard white light visualization used in clinicalexamination of the wounds. Inset b) of FIG. 18 shows a correspondingfluorescence image of the same wound (405 nm excitation, 500-550 nmemission (green), >600 nm emission (red)). Fluorescence imaging showsthe presence of bacteria around the wound edge/border pre-cleaning(inset (b)) and post-cleaning (inset (c) of FIG. 18 ). In this example,cleaning involved the use of standard gauze and phosphate bufferedsaline to wipe the surface of the wound (within and without) for 5minutes. After cleaning, the red fluorescence of the bacteria isappreciably decreased indicating that some of the red fluorescentbacteria may reside below the tissue surface around the edge of thewound. Small amounts of bacteria (red fluorescent) remained within thewound center after cleaning. This illustrates the use of the imagingdevice to monitor the effects of wound cleaning in real-time. As anadditional example, inset d) of FIG. 18 shows a white light image of achronic non-healing wound in the same patient located on the left calf.Inset e) of FIG. 18 shows the corresponding fluorescence imagepre-cleaning (inset (e)) and post-cleaning (inset (f) of FIG. 18 ).Swabbing of the central area of the wound revealed the occasional growthof Staphylococcus aureus, with a heavy growth of Staphylococcus aureusat the edge (yellow arrow). Cleaning resulted in a reduction of thefluorescent bacteria (Staphylococcus aureus) on the wound surface asdetermined using the handheld optical imaging device. The use of theimaging device resulted in the real-time detection of white light-occultbacteria and this allowed an alteration in the way the patient wastreated such that, following fluorescence imaging, wounds andsurrounding (bacteria contaminated) were either re-cleaned thoroughly orcleaned for the first time because of de novo detection of bacteria.Also, note that the use of a disposable adhesive measurement-calibration‘strip’ for aiding in imaging-focusing and this “strip” may be adheredto any part of the body surface (e.g., near a wound) to allow woundspatial measurements. The calibration strip may also be distinctlyfluorescent and may be used to add patient-specific information to theimages, including the use of multiple exogenous fluorescent dyes for“barcoding” purposes—the information of which can be integrated directlyinto the fluorescence images of wounds. (Scale bar in cm).

FIG. 19 illustrates use of an imaging devices in accordance with thepresent disclosure for monitoring wound healing over time. The imagingdevice of the present disclosure is used for tracking changes in thehealing status and bacterial biodistribution (e.g. contamination) of anon-healing chronic wound from the left breast of a female patient withPyoderma gangrenosum. White light images (see column showing insets a-min FIG. 19 ) and corresponding fluorescence images of the healed wound(see column showing insets b-n in FIG. 19 ) and of the chronicnon-healing wound (see column showing insets c-o in FIG. 19 ) are shownover the course of six weeks. (405 nm excitation, 500-550 nm emission(green), >600 nm emission (red)), taken using the imaging device underboth white light and fluorescence modes. In the column of insets b-n,the presence of small bright red fluorescence bacterial colonies isdetected (yellow arrows), and their localization changes over timewithin the healed wound. Bacterial swabs confirmed that no bacteria weredetected on microscopy and no bacterial growth was observed in culture.In the column of insets c-o), by contrast, the non-healed wound has aband of very bright red fluorescence around the wound border, confirmedwith swab cultures (bacteriology) to contain a heavy growth ofStaphylococcus aureus (with few Gram positive bacilli and rare Grampositive cocci, confirmed by microscopy), which changes inbiodistribution over time (i.e., see column of insets c-o). This datademonstrates that imaging devices in accordance with the presentdisclosure may yield real-time biological and molecular information aswell as be used to monitor morphological and molecular changes in woundsover time.

FIG. 20 shows another example of the use of the devices in accordancewith the present disclosure for monitoring wound status over time. InFIG. 20 , the imaging device was used to track changes in the healingstatus and bacterial biodistribution (e.g. contamination) of a woundfrom the left calf of 21 year old female patient with Pyodermagangrenosum. White light images (see column of insets a-i in FIG. 20 )and corresponding fluorescence images (see column of insets b-j in FIG.20 ) of a wound being treated using hyperbaric oxygen therapy (HOT) areshown over the course of six weeks. (Fluorescence parameters: 405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)). Columnof insets a-i) White light images reveal distinct macroscopic changes inthe wound as it heals, indicated by the reduction in size over time(e.g. closure) from week 1 (˜2 cm long axis diameter) through to week 6(˜0.75 cm long axis diameter). In the column of insets b-j, thereal-time fluorescence imaging of endogenous bacterial fluorescence(autofluorescence) in and around the wound can be tracked over time andcorrelated with the white light images and wound closure measurements(column of insets a-i). Inset b of FIG. 20 shows a distinct green bandof fluorescence at the immediate boundary of the wound (yellow arrow;shown to be contaminated heavy growth of Staphylococcus aureus), andthis band changes over time as the wound heals. Red fluorescencebacteria are also seen further away from the wound (orange arrow), andtheir biodistribution changes over time (see column of insets b-j). Thewound-to-periwound-to-normal tissue boundaries can be seen clearly byfluorescence in image inset j of FIG. 20 . Connective tissue (in thisexample, collagens) in normal skin appear as pale green fluorescence(inset j) and connective tissue remodeling during wound healing can bemonitored over time, during various wound treatments including, as isthe case here, hyperbaric oxygen therapy of chronic wounds.

FIG. 21 illustrates use of imaging devices in accordance with thepresent disclosure for targeting bacterial swabs during routine woundassessment in the clinic. Under fluorescence imaging, the swab can bedirected or targeted to specific areas of bacterialcontamination/infection using fluorescence image-guidance in real-time.This may decrease the potential for contamination of non-infectedtissues by reducing the spread of bacteria during routine swabbingprocedures, which may be a problem in conventional wound swabbingmethods. Swab results from this sample were determined to beStaphylococcus aureus (with few Gram positive bacilli and rare Grampositive cocci, confirmed by microscopy).

FIG. 22 shows an example of the co-registration of a) white light and b)corresponding fluorescence images made with an imaging device inaccordance with the present disclosure in a patient withdiabetes-associated non-healing foot ulcers. Using a non-contacttemperature measuring probe (inset in a) with cross-laser sighting,direct temperature measurements were made on normal skin (yellow “3 and4”) and within the foot ulcers (yellow “1 and 2”) (infected withPseudomonas aeruginosa, as confirmed by bacteriological culture),indicating the ability to add temperature-based information to the woundassessment during the clinical examination. Infected wounds haveelevated temperatures, as seen by the average 34.45° C. in the infectedwounds compared with the ° C. on the normal skin surface, and this dataillustrates the possibility of multimodality measurements, which includewhite light, fluorescence and thermal information for woundhealth/infectious assessment in real-time. Note that both non-healingwounds on this patient's right foot contained heavy growth ofPseudomonas aeruginosa (in addition to Gram positive cocci and Gramnegative bacilli), which in this example appear as bright greenfluorescent areas within the wound (inset b) of FIG. 22 ).

FIG. 23 shows an example of the use of an imaging device in accordancewith the present disclosure for monitoring a pressure ulcer. Inset a ofFIG. 23 shows a white light image taken with the imaging device inaccordance with the present disclosure of the right foot of a Caucasiandiabetic patient with a pressure ulcer. Inset b of FIG. 23 shows acorresponding fluorescence image showing the bright red fluorescence ofbacteria (bacteriology results confirmed presence of heavy growth ofStaphylococcus aureus), which are invisible under standard white lightexamination (yellow arrows). Dead skin appears as a white/pale lightgreen color (white arrows). Note the heavy growth of Staphylococcusaureus bacteria around the periphery of the non-healing open wounds(yellow arrows). Inset c) of FIG. 23 shows the fluorescence imaging of atopically applied silver antimicrobial dressing. The imaging device inaccordance with the present disclosure may be used to detect theendogenous fluorescence signal from advanced wound care products (e.g.,hydrogels, wound dressings, etc.) or the fluorescence signals from suchproducts that have been prepared with a fluorescent dye with an emissionwavelength within the detection sensitivity of the imaging detector onthe device. The device may be used for image-guided delivery/applicationof advanced wound care treatment products and to subsequently monitortheir distribution and clearance over time.

FIG. 24 shows another example of the use of a device in accordance withthe present disclosure for monitoring a pressure ulcer. Inset a) of FIG.24 shows a white light image taken with the device of the presentdisclosure of the right foot of a Caucasian diabetic patient with apressure ulcer. Inset b) of FIG. 24 shows a corresponding fluorescenceimage showing the bright red fluorescent area of bacteria (bacteriologyresults confirmed presence of heavy growth of Staphylococcus aureus, SA)at the wound edge and bright green fluorescent bacteria (bacteriologyresults confirmed presence of heavy growth of Pseudomonas aeruginosa,PA) which are both invisible under standard white light examination.Inset c) of FIG. 24 shows fluorescence spectroscopy taken of the woundthat revealed unique spectral differences between these two bacterialspecies: SA has a characteristic red (about 630 nm) autofluorescenceemission peak, while PA lacks the red fluorescence but has a stronggreen autofluorescence peak at around 480 nm.

The handheld device in accordance with the present disclosure spectrallydistinguishes bacteria from connective tissues and blood in vivo. Usingλexc=405_20 nm and λemiss=500 to 550 nm, 590 to 690 nm, the devicedetects AF signals of S. aureus, Staphylococcus epidermidis, P.aeruginosa, Candida, Serratia marcescens, Viridans streptococci(α-hemolytic streptococci), Streptococcus pyogenes (β-hemolyticstreptococci), Corynebacterium diphtheriae, Enterobacter, Enterococcus,and methicillin-resistant S. aureus (MRSA), as verified bymicrobiological swab cultures (data from a human clinical trial by ourgroup to be published in a forthcoming paper). This is a representativeof the major types of pathogenic bacteria commonly found in infectedwounds. Clinical microbiology tests confirmed that S. aureus, S.epidermidis, Candida, S. marcescens, Viridans streptococci,Corynebacterium diphtheriae, S. pyogenes, Enterobacter, and Enterococcusproduced red FL (from porphyrin) while P. aeruginosa produced abluish-green FL (from pyoverdin) detected by the handheld device. Thesespectral characteristics differ significantly from connective tissues(collagen, elastin) and blood, which appear green and dark red,respectively. A representative image of these spectral characteristicsis shown in FIG. 24 .

FIG. 25 shows an example of the use of a device in accordance with thepresent disclosure for monitoring a chronic non-healing wound. Inset a)of FIG. 25 shows a white light image taken with the imaging device ofthe present disclosure of chronic non-healing wounds in a 44 year oldblack male patient with Type II diabetes. Bacteria cannot be visualizedby standard white light visualization (see column of insets a-g) of FIG.25 ) used in conventional clinical examination of the wounds. Column ofinsets b-h) of FIG. show corresponding fluorescence images of the samewounds (405 nm excitation, 500-550 nm emission (green), >600 nm emission(red)). This patient presented with multiple open non-healing wounds.Swab cultures taken from each wound area using the fluorescenceimage-guidance revealed the heavy growths of Pseudomonas aeruginosa(yellow arrow), which appear bright green fluorescent, and Serratiamarcescens (circles), which appear red fluorescent. (Scale bar in cm).

FIG. 26 is a schematic diagram illustrating an example of a use of“calibration” targets, which may be custom-designed, multi-purpose,and/or disposable, for use during wound imaging with imaging devices inaccordance with the present disclosure. The strip, which in this exampleis adhesive, may contain a combination of one or more of: spatialmeasurement tools (e.g., length scale), information barcode forintegrating patient-specific medical information, and impregnatedconcentration-gradients of fluorescent dyes for real-time fluorescenceimage calibration during imaging. For the latter, multipleconcentrations of various exogenous fluorescent dyes or otherfluorescence agents (e.g., quantum dots) may be used for multiplexedfluorescence intensity calibration, for example when more than oneexogenous fluorescently-labeled probe is used fortissue/cell/molecular-targeted molecular imaging of wounds in vivo.

FIG. 27 shows an example of the use of an embodiment of the imagingdevice for monitoring bacteria, for example for monitoring a treatmentresponse. Inset a) of FIG. 27 shows a fluorescence microscopy image of alive/dead bacteria stain sold by Invitrogen Corp. (i.e., BacLightproduct). Inset b) of FIG. 27 shows a fluorescence microscopy image of aGram staining bacteria labeling stain sold by Invitrogen Corp. Using theimaging device, as shown in inset c) of FIG. 27 , with such products,live (green) and dead (red) bacteria may be distinguished in real-timeex vivo (e.g., on the swab or tissue biopsy shown in inset e of FIG. 27) following bacterial swabbing of a wound, or other body surface, forexample, in the swabbing of the oral buccal cheek, as in inset d of FIG.27 . This real-time bacterial Gram staining or live/dead image-basedassessment may be useful for real-time or relatively rapid bacteriologyresults that may be used for refining treatments, such as antibiotic orother disinfective treatments, or for monitoring treatment response.

FIG. 28 shows an example of the use of a device in accordance with thepresent disclosure used for imaging of a toe nail infection. Inset a) ofFIG. 28 shows a white light image and inset b) of FIG. 28 shows acorresponding autofluorescence image of the right toe of a subjectdemonstrating the enhanced contrast of the infection that fluorescenceimaging provides compared to white light visualization (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)).

ADDITIONAL EXAMPLES

FIG. 29 shows an example of a device in accordance with the presentdisclosure being used for non-invasive autofluorescence detection ofcollagen and varies bacterial species on the skin surface of a pig meatsample. In contrast to white light imaging, autofluorescence imaging wasable to detect the presence of several bacterial species 24 hours afterthey were topically applied to small incisions made in the skin (i.e.,Streptococcus pyogenes, Serratia marcescens, Staphylococcus aureus,Staphylococcus epidermidis, Escherichia coli, and Pseudomonasaeruginosa). Inset a) of FIG. 29 shows a white light image of pig meatused for testing. Several bacterial species were applied to smallincisions made in the skin at Day 0 and were labelled as follows: 1)Streptococcus pyogenes, 2) Serratia marcescens, 3) Staphylococcusaureus, 4) Staphylococcus epidermidis, 5) Escherichia coli, and 6)Pseudomonas aeruginosa. The imaging device was used to detect collagenand bacterial autofluorescence over time. Connective tissue fluorescencewas intense and easily detected as well. Some bacterial species (e.g.,Pseudomonas aeruginosa) produces significant green autofluorescence(450-505 nm) which saturated the device's camera. Inset b) of FIG. 29shows an autofluorescence image at Day 0, magnified in inset c) of FIG.29 .

The device was also able to detect spreading of the bacteria over thesurface of the meat over time. Inset d) of FIG. 29 shows an image at Day1, and inset f) of FIG. 29 shows an image at Day 3, as the meat samplewas maintained at 37° C. Red fluorescence can be seen in some of thewound sites (5, 6) in inset c) of FIG. 29 . As shown in inset d) andmagnified in inset e) of FIG. 29 , after 24 h, the device detects adramatic increase in bacterial autofluorescence from wound site 5)Escherichia coli and 6) Pseudomonas aeruginosa, with the latterproducing significant green and red autofluorescence. Insets c) and e)of FIG. 29 show the device detecting fluorescence using a dual band(450-505 nm green and 590-650 nm) on the left and a single band filter(635+/−10 nm) on the right, of the wound surface. As shown in inset f),by Day 3, the device detects the significant increase in bacterialautofluorescence (in green and red) from the other wound sites, as wellas the bacterial contamination (indicated by the arrow in inset f) onthe styrofoam container in which the meat sample was kept. The devicewas also able to detect spreading of the bacteria over the surface ofthe meat. This demonstrates the real-time detection of bacterial specieson simulated wounds, the growth of those bacteria over time, and thecapability of the device to provide longitudinal monitoring of bacterialgrowth in wounds. The device may provide critical information on thebiodistribution of the bacteria on the wound surface which may be usefulfor targeting bacterial swabbing and tissue biopsies. Note, in insets d)and f), the intense green fluorescence signal from endogenous collagenat the edge of the pig meat sample.

This example demonstrates the use of devices in accordance with thepresent disclosure for real-time detection of biological changes inconnective tissue and bacterial growth based on autofluorescence alone,suggesting a practical capability of the device to provide longitudinalmonitoring of bacterial growth in wounds.

Referring again to FIG. 3 , the images show examples of a device inaccordance with the present disclosure used for autofluorescencedetection of connective tissues (e.g., collagen, elastin) and bacteriaon the muscle surface of a pig meat sample. Inset a) of FIG. 3 showsthat white light image of pig meat used for testing shows no obvioussigns of bacterial/microbial contamination or spoilage. However, as seenin inset b) of FIG. 3 , imaging of the same area with the device underblue/violet light excitation revealed a bright red fluorescent area ofthe muscle indicating the potential for bacterial contamination comparedwith the adjacent side of muscle. Extremely bright greenautofluorescence of collagen can also be seen at the edge of the skin.In inset c) of FIG. 3 , the device was used to surgically interrogatesuspicious red fluorescence further to provide a targeted biopsy forsubsequent pathology or bacteriology. Note also the capability of thedevice to detect by fluorescence the contamination (arrow) of thesurgical instrument (e.g., forceps) during surgery. In inset d) of FIG.3 , the device was used to target the collection of fluorescencespectroscopy using a fibre optic probe of an area suspected to beinfected by bacteria (inset shows the device being used to target thespectroscopy probe in the same area of red fluorescent muscle in insetsb) and c). Inset e) of FIG. 3 shows an example of the device being usedto detect contamination by various thin films of bacteria on the surfaceof the Styrofoam container on which the meat sample was kept.Autofluorescence of the bacteria appears as streaks of green and redfluorescence under violet/blue excitation light from the variousbacterial species previously applied to the meat. Thus, the device iscapable of detecting bacteria on non-biological surfaces where they areoccult under standard white light viewing (as in inset a)).

In addition to detection of bacteria in wounds and on the skin surface,the device was also able to identify suspicious areas of muscle tissue,which may then be interrogated further by surgery or targeted biopsy forpathological verification, or by other optical means such asfluorescence spectroscopy using a fiber optic probe. Also, it detectedcontamination by various bacteria on the surface of the Styrofoamcontainer on which the meat sample was kept. Autofluorescence of thebacteria appears as streaks of green and red fluorescence underviolet/blue excitation light from the various bacterial speciespreviously applied to the meat.

In order to determine the autofluorescence characteristics of bacteriagrowing in culture and in simulated skin wounds,hyperspectral/multispectral fluorescence imaging was used toquantitatively measure the fluorescence intensity spectra from thebacteria under violet/blue light excitation. Reference is now made toFIG. 30 . In FIG. 30 , a device in accordance with the presentdisclosure was used to detect fluorescence from bacteria growing in agarplates and on the surface of a simulated wound on pig meat, as discussedabove for FIGS. 12 and 29 . Bacterial autofluorescence was detected inthe green and red wavelength ranges using the device in the culture(inset a) of FIG. 30 ) and meat samples (inset d) of FIG. 30 ).Hyperspectral/multispectral imaging was used to image the bacteria (E.Coli) in culture (inset b) of FIG. 30 ) and to measure the quantitativefluorescence intensity spectra from the bacteria (red line—porphyrins,green—cytoplasm, blue—agar background) (inset c) of FIG. 30 ). The redarrow shows the 635 nm peak of porphyrin fluorescence detected in thebacteria. Hyperspectral/multispectral imaging also confirmed the stronggreen fluorescence (*, right square in inset d) from P. aeruginosa (withlittle porphyrin fluorescence, yellow line in inset f) of FIG. 30compared to E. coli (left square in inset d) where significant porphyrinred fluorescence was detected. Insets e) and g) of FIG. 30 show thecolor-coded hyperspectral/multispectral images corresponding to P.aeruginosa and E. coli, respectively, from the meat surface after 2 daysof growth (incubated at 37° C.); and insets f) and h) of FIG. 30 showthe corresponding color-coded fluorescence spectroscopy. In inset i) ofFIG. 30 , excitation-emission matrices (EEM) were also measured for thevarious bacterial species in solution, demonstrating the ability toselect the optimum excitation and emission wavelength bandwidths for usewith optical filters in the imaging device. The EEM for E. coli showsstrong green fluorescence as well as significant red fluorescence fromendogenous bacterial porphyrins (arrow).

This example shows that bacteria emit green and red autofluorescence,with some species (e.g., Pseudomonas aeruginosa) producing more of theformer. Escherichia coli produced significant red autofluorescence fromendogenous porphyrins. Such intrinsic spectral differences betweenbacterial species are significant because it may provide a means ofdifferentiating between different bacterial species usingautofluorescence alone. Excitation-emission matrices (EEMs) were alsomeasured for each of the bacterial species used in these pilot studies,which confirmed that under violet/blue light excitation, all speciesproduced significant green and/or red fluorescence, the latter beingproduced by porphyrins. Spectral information derived fromexcitation-emission matrices may aid in optimizing the selection ofexcitation and emission wavelength bandwidths for use with opticalfilters in the imaging device to permit inter-bacterial speciesdifferentiating ex vivo and in vivo. In this way, the device may be usedto detect subtle changes in the presence and amount of endogenousconnective tissues (e.g. collagens and elastins) as well as bacteriaand/or other microorganisms, such as yeast, fungus and mold withinwounds and surrounding normal tissues, based on unique autofluorescencesignatures of these biological components.

This device may be used as an imaging and/or monitoring device inclinical microbiology laboratories. For example, the device may be usedfor quantitative imaging of bacterial colonies and quantifying colonygrowth in common microbiology assays. Fluorescence imaging of bacterialcolonies may be used to determine growth kinetics.

Imaging of Blood in Wounds

Angiogenesis, the growth of new blood vessels, is an important naturalprocess required for healing wounds and for restoring blood flow totissues after injury or insult. Angiogenesis therapies, which aredesigned to “turn on” new capillary growth, are revolutionizing medicineby providing a unified approach for treating crippling andlife-threatening conditions. Angiogenesis is a physiological processrequired for wound healing. Immediately following injury, angiogenesisis initiated by multiple molecular signals, including hemostaticfactors, inflammation, cytokine growth factors, and cell-matrixinteractions. New capillaries proliferate via a cascade of biologicalevents to form granulation tissue in the wound bed. This process may besustained until the terminal stages of healing, when angiogenesis ishalted by diminished levels of growth factors, resolution ofinflammation, stabilized tissue matrix, and endogenous inhibitors ofangiogenesis. Defects in the angiogenesis pathway impair granulation anddelay healing, and these are evident in chronic wounds. By illuminatingthe tissue surface with selected narrow wavelength bands (e.g., blue,green and red components) of light or detecting the reflectance of whitelight within several narrow bandwidths of the visible spectrum (e.g.,selected wavelengths of peak absorption from the blood absorptionspectrum of white light), the device may also be used to image thepresence of blood and microvascular networks within and around thewound, including the surrounding normal tissue, thus also revealingareas of erythema and inflammation.

Reference is now made to FIG. 31 . In this example, a device inaccordance with the present disclosure may use individual opticalfilters (e.g., 405 nm, 546 nm, 600 nm, +/−25 nm each) in order todemonstrate the possibility of imaging blood and microvasculature inwounds. White light images of a wound may be collected with the deviceand then the device, equipped with a triple band-pass filter (e.g., 405nm, 546 nm, 600 nm, +/−25 nm each), placed in front of the imagingdetector may image the separate narrow bandwidths of blue (B), green(G), and red (R) reflected light components from the wound. Thesewavelength bands may be selected based on the peak absorptionwavelengths of blood, containing both oxygenated and deoxygenatedhemoglobin, in the visible light wavelength range. The resulting imagesmay yield the relative absorption, and thus reflectance, of visiblelight by blood in the field of view. The resulting ‘blood absorption’image yields a high contrast image of the presence of blood and/ormicrovascular networks in the wound and surrounding normal tissues. Theclinician may select the appropriate optical filter set for use with thedevice to obtain images of blood and/or microvascular distributionwithin the wound and then combine this information with one or both ofautofluorescence imaging and imaging with exogenous contrast agents.This may provide a comprehensive information set of the wound andsurrounding normal tissues at the morphological, topographical,anatomical, physiological, biological and molecular levels, whichcurrently may not be possible within conventional wound care practice.

FIG. 31 shows examples of the device used for imaging of blood andmicrovasculature in wounds. The device was used to image a piece offilter paper stained with blood (inset a) of FIG. 31 ) and the ear of amouse during surgery (inset b) of FIG. 31 ). White light images werecollected of each specimen using the imaging device, in non-fluorescencemode, and then the device was equipped with a triple band-pass filterplaced in front of the imaging detector (405 nm, 546 nm, 600 nm, +/−25nm each) to image the separate narrow bandwidths of blue (B), green (G),and red (R) reflected light components from the specimens. Thesewavelength bands were selected based on the peak absorption wavelengthsof blood in the visible light wavelength range. Inset a) shows theabsorption spectral profile for oxy- and deoxygenated hemoglobin inblood. This shows that when using a simple multiband transmissionfilter, it is possible to combine the three B, G, R images into a single‘white light equivalent’ image that measures the relative absorption oflight by blood in the field of view. The resulting ‘blood absorption’image yields a high contrast image of the presence of blood containingboth oxy- and deoxygenated hemoglobin. The device may also be used withnarrower bandwidth filters to yield higher contrast images of bloodabsorption in wounds, for example.

The regulation of angiogenesis over time during wound repair in vivo hasbeen largely unexplored, due to difficulties in observing events withinblood vessels. Although initial tests using an imaging device inaccordance with the present disclosure were exploratory, simplemodification of the device may allow longitudinal imaging of dynamicchanges in blood supply and microvascular networks during the woundhealing process in vivo.

In general, devices in accordance with the present disclosure may beused to image and/or monitor targets such as a skin target, an oraltarget, an ear-nose-throat target, an ocular target, a genital target,an anal target, and any other suitable targets on a subject.

Use in Clinical Care

Although current wound management practice aims to decrease themorbidity and mortality of wounds in patients, a limitation is theavailability of health care resources. The potential of incorporatingthe technology of telemedicine into wound care needs is currently beingexplored. Wound care is a representation of the care of chronic anddebilitating conditions that require long-term specialized care. Themajor effect of improved living conditions and advances in health careglobally has led to people living longer. Therefore, the percentage ofworlds' elderly and those with chronic medical conditions that wouldrequire medical attention is rising. With the escalating costs of healthcare, and the push of the industry towards outpatient care, this is apart of the health care crisis that is demanding immediate attention.

Devices in accordance with the present disclosure may providebiologically-relevant information about wounds and may exploit theemerging telemedicine (e.g., E-health) infrastructure to provide asolution for mobile wound care technology and may greatly impact woundhealth care treatment. Wound care accounts for a large percentage ofhome visits conducted by nurses and health care workers. Despite bestpractices some wounds do not heal as expected and require the servicesof a clinical specialist. The exemplary devices described herein mayenable access to specialized clinical resources to help treat woundsfrom the convenience of the patient's home or chronic care facility,which decreases travel time for clients, increases availability toclinical wound specialists, and may reduce costs to the health caresystem.

Different uses of the imaging devices have been discussed for woundassessment, monitoring and care management. The devices may be used todetect and monitor changes in connective tissues (e.g., collagen,elastin) and blood/vascular supply during the wound healing process,monitor tissue necrosis and exudate in wounds based on fluorescence,detect and diagnose wound infections including potentially indicatingcritical ‘clinically significant’ categories of the presence of bacteriaor micro-organisms (e.g., for detecting contamination, colonization,critical colonization and infection) at the surface and deep withinwounds, provide topographic information of the wound, and identify woundmargins and surrounding normal tissues. Tissue fluorescence andreflectance imaging data may be ‘mapped’ onto the white light images ofthe wound thereby permitting visualization within the wound and thesurrounding normal tissues of essential wound biochemical andphotobiological (e.g., fluorescence) information, which has not beenpossible to date. Real-time imaging of wounds may be performed over timeto monitor changes in wound healing, and to potentially monitor theeffectiveness of treatments by providing useful information aboutunderlying biological changes that are occurring at the tissue/cellularlevel (e.g., matrix remodeling, inflammation, infection and necrosis).This may provide quantitative and objective wound information fordetection, diagnosis and treatment monitoring in patients. Inparticular, such devices may be used to monitor and/or track theeffectiveness of therapy at a biological level (e.g., on a bacteriallevel), which may provide more information than monitoring only themacroscopic/morphological appearance using white light.

The devices may provide real-time non-invasive image-guided biopsytargeting, clinical procedural guidance, tissue characterization, andmay enable image-guided treatment using conventional and emergingmodalities (e.g., PDT). In addition, use of the imaging devices may beused to correlate critical biological and molecular wound informationobtained by fluorescence (e.g., endogenous tissue autofluorescenceand/or administration of exogenous molecular-biomarker targetedfluorescence contrast agents) with existing and emerging clinical woundcare assessment and treatment guides, such as the NERDS and STONESguidelines proposed by Sibbald et al. (Sibbald et al. IncreasedBacterial Burden and Infection: The Story of NERDS and STONES. ADV SKINWOUND CARE 2006; 19:447-61). The fluorescence imaging data obtained withthe devices may be used to characterize, spatially and spectrally,bacterial balance and burden at the superficial and deep levels ofwounds. The devices may provide real-time non-invasive image-guidedbiopsy targeting, clinical procedural guidance, tissue characterization,and may enable image-guided treatment using conventional and emergingtreatment modalities (e.g., photodynamic therapy, PDT). The devices maybe used within the clinical setting and integrated into conventionalclinical wound care regimens, and may have a distinct role in areas ofinfectious diseases. It should be noted as well that such devices mayalso be used for real-time analysis, monitoring and care for chronic andacute wounds in animals and pets, via conventional veterinary care.

Devices in accordance with the present disclosure may allow real-timewound healing assessment for a large patient cohort base. In particular,elderly people, diabetics, immuno-suppressed and immobilized individualshave an increased incidence of chronic wounds and other dermalafflictions that result from poor circulation and immobility, e.g.pressure ulcers such as bed sores, venous stasis ulcers, and diabeticulcers. These chronic conditions greatly increase the cost of care andreduce the patient's quality of life. As these groups are growing innumber, the need for advanced wound care products will increase. Suchdevices may impact patient care by allowing a cost-effective means ofmonitoring chronic and acute wounds in a number of settings, includinghospitals, ambulatory clinics, chronic care facilities, in-home-visithealth care, emergency rooms and other critical areas in health carefacilities. Further, such ‘handheld’ and portable imaging devices may beeasily carried and used by nursing and ambulance staff. Earlyidentification of scarring, which is related to connective tissueproduction and remodeling of the wound, and bacterial infections may bedetected and treated appropriately, something that is currentlydifficult. In addition, recent developments in advanced wound-careproducts including multiple dressing types (e.g., film, hydrocolloid,foam, anti-microbial, alginate, non-adherent, impregnated), hydrogels,wound cleansers and debriding agents, tissue engineered products (e.g.,skin replacements, substitutes, and tissue-engineered products such assynthetic polymer-based biological tissue and growth factors), woundcleansers, pharmacological products, and physical therapies may alsobenefit from the device developed here as it may allow image-basedlongitudinal monitoring of the effectiveness of such treatments.Physical therapies may include hydrotherapy, electrical stimulation,electromagnetic stimulation devices, ultraviolet therapy, hyperbaricoxygen therapy, ultrasound devices, laser/light emitting diode (LED)devices, and wound imaging/documentation. Additional therapies mayinclude, for example, antibiotics, wound debridement, application ofwound dressings, and wound cleaning.

Wound tissue analysis is typically required for the assessment of thehealing of skin wounds. Percentage of the granulation tissue, fibrin andnecrosis in the wound, and their change during treatment may provideuseful information that may guide wound treatment. Image analysis mayinclude advanced statistical pattern recognition and classificationalgorithms to identify individual pixels within the fluorescence woundimages collected with the device based on the optical information of thewound and surrounding normal tissue. Thus, image analysis may allowwound images to be mapped into various components of the wound,including total wound area, epithelialization, granulation, slough,necrotic, hypergranulation, infected, undermining, and surroundingtissue margins. This has an added advantage of providing relativelyrapid determination of wound healing rates, as well as informing guidepatient management decisions.

FIG. 32 illustrates the projected management workflow for an exemplaryimaging device in a clinical wound care setting. The device may beeasily integrated into routine wound assessment, diagnosis, treatmentand longitudinal monitoring of response, and may provide criticalbiological and molecular information of the wound in real-time for rapiddecision-making during adaptive interventions.

This device may be easily integrated into existing health-care computerinfrastructures (e.g., desktop and pocket PCs used by a growing numberof physicians or other health care professionals) for longitudinal imagecataloguing for patient wound management within the conventionalclinical environment. The wireless receiving and transmission of datacapabilities of the device may allow monitoring of wound care andhealing remotely through existing and future wireless telemedicineinfrastructure. The device may be used to transfer essential medicaldata (e.g., wound health status) via the internet or over wirelessservices, such as cellular telephone, PDA or Smartphone services, toremote sites which may permit remote medical interventions, with afurther utility in military medical applications for battlefield woundmanagement. The device may allow real-time surface imaging of woundsites and may be easily carried by point-of-care personnel in clinicalsettings. Using cost-effective highly sensitive commercially availabledigital imaging devices, such as digital cameras, cellular phones, PDAs,laptop computers, tablet PCs, webcams, and Smart phones, etc. as theimage capture or recording component, the device may offer image-baseddocumentation of wound healing and tracking of treatment effectiveness.Also, this technology may be adapted to also function in ‘wireless’ modeto permit remote medical interventions by potentially adapting it foruse with high-resolution digital cameras embedded incommercially-available cellular telephones.

By using web-based telemedicine and remote medical monitoringinfrastructure, the imaging device may be integrated into a‘store-and-forward’ concept of wound assessment systems. In addition toproviding digital images, such a system may present a comprehensive setof clinical data that meet the recommendations of clinical practiceguidelines. The presently-disclosed devices may integrate into acomputer-based wound assessment system (e.g., with image analysissoftware) to be used by a health care facility to enhance existingclinical databases and support the implementation of evidence—basedpractice guidelines. Such an integrated telemedicine infrastructure maybe used for monitoring patients at home or in long-term-care facilities,who may benefit from routine monitoring by qualified clinicians butcurrently do not have access to this care. These devices may be furtherdeveloped into a portable handheld point-of-care diagnostic system,which may represent a major advance in detecting, monitoring, treating,and preventing infectious disease spread in the developed and developingworlds. This knowledge may significantly improve the diagnostic toolsavailable to practitioners who treat chronic wounds in settings wherequantitative cultures are inaccessible.

Devices in accordance with the present disclosure may allow digitalimaging with optical and digital zooming capabilities (e.g., thoseembedded in commonly available digital imaging devices). Still or videoimage quality may be in ‘high-definition’ format to achieve high spatialresolution imaging of the tissue surface. Images may be recorded asstill/freeze frame and/or in video/movie format and printed usingstandard imaging printing protocols which do (e.g., connected via USB)or do not (e.g., PictBridge) require a personal computer. Theimages/video data may be transferred to a personal computer for dataarchival storage and/or image viewing and/or analysis/manipulation. Suchdevices may also transfer data to a printer or personal computer usingwired or wireless capabilities (e.g., Bluetooth). Visualization may beperformed on the handheld device screen and/or in addition tosimultaneous viewing on a video screen/monitor (e.g., head-mounteddisplays and glasses) using standard output video cables. These devicesmay display, in combination or separately, optical wavelength andfluorescence/reflectance intensity information with spatial dimensionsof the imaged scene to allow quantitative measurements of distances(e.g., monitoring changes tissue morphology/topography) over time. Thedevices may also allow digital image/video storage/cataloguing of imagesand related patient medical data, for example using dedicated softwarewith imaging analysis capabilities and/or diagnostic algorithms.

Image Analysis

Image analysis may be used together with the exemplary devices of thepresent disclosure to quantitatively measure fluorescence intensitiesand relative changes in multiple fluorescence spectra (e.g., multiplexedimaging) of the exogenous optical molecular targeting probes in thewound and surrounding normal tissues. The biodistributions of thefluorescent probes may be determined based on the fluorescence imagescollected and these may be monitored over time between individualclinical wound imaging sessions for change. By determining the presenceand relative changes in abundance quantitatively, using the devices, ofeach and all of the spectrally-unique fluorescent probes, the clinicaloperator may determine in real-time or near real-time the health and/orhealing status and response to treatment over time of a given wound, forexample by using a look-up table in which specific tissue, cellular andmolecular signals are displayed in correlation to wound health, healingand response status, an example of which is shown in FIG. 33 . This maypermit the clinician to determine whether a wound is healing based onbiological and molecular information which may not be possible otherwisewith existing technologies. Furthermore, the presence and abundance ofbacteria/microorganisms and their response to treatment may offer ameans to adapt the therapy in real-time instead of incurring delays inresponse assessment with conventional bacteriological testing of woundcultures.

Image analysis techniques may be used to calibrate the initial or firstimages of the wound using a portable fluorescent standard placed withinthe field of view during imaging with a device. The image analysis mayalso permit false or pseudo color display on a monitor fordifferentiating different biological (e.g., tissue, cellular, andmolecular) components of the wound and surrounding normal tissuesincluding those biomarkers identified by autofluorescence and thoseidentified by the use of exogenous targeted or untargetedfluorescence/absorption contrast agents.

Examples of such biomarkers are listed in FIG. 34 and illustrated inFIG. 35 . In FIG. 35 , the diagram shows mechanisms of wound healing inhealthy people versus people with diabetic wounds. In healthyindividuals (left side of FIG. 35 ), the acute wound healing process isguided and maintained through integration of multiple molecular signals(e.g., in the form of cytokines and chemokines) released bykeratinocytes, fibroblasts, endothelial cells, macrophages, andplatelets. During wound-induced hypoxia, vascular endothelial growthfactor (VEGF) released by macrophages, fibroblasts, and epithelial cellsinduces the phosphorylation and activation of eNOS in the bone marrow,resulting in an increase in NO levels, which triggers the mobilizationof bone marrow EPCs to the circulation. For example, the chemokineSDF-lapromotes the homing of these EPCs to the site of injury, wherethey participate in neovasculogenesis. In a murine model of diabetes(right side of FIG. 35 ), eNOS phosphorylation in the bone marrow isimpaired, which directly limits EPC mobilization from the bone marrowinto the circulation. SDF-1 α expression is decreased in epithelialcells and myofibroblasts in the diabetic wound, which prevents EPChoming to wounds and therefore limits wound healing. It has been shownthat establishing hyperoxia in wound tissue (e.g., via HBO therapy)activated many NOS isoforms, increased NO levels, and enhanced EPCmobilization to the circulation. However, local administration of SDF-1α was required to trigger homing of these cells to the wound site. Theseresults suggest that HBO therapy combined with SDF-1 α administrationmay be a potential therapeutic option to accelerate diabetic woundhealing alone or in combination with existing clinical protocols.

Pre-assigned color maps may be used to display simultaneously thebiological components of the wound and surrounding normal tissuesincluding connective tissues, blood, microvascularity, bacteria,microorganisms, etc. as well as fluorescently labeleddrugs/pharmacological agents. This may permit visualization in real-timeor near real-time (e.g., less than 1 minute) of the health, healing andinfectious status of the wound area.

The image analysis algorithms may provide one or more of the followingfeatures:

Patient Digital Image Management

-   -   Integration of a variety of image acquisition devices    -   Records all imaging parameters including all exogenous        fluorescence contrast agents    -   Multiple scale and calibrations settings    -   Built-in spectral image un-mixing and calculation algorithms for        quantitative determination of tissue/bacterial autofluorescence        and exogenous agent fluorescence signals    -   Convenient annotation tools    -   Digital archiving    -   Web publishing

Basic Image Processing and Analysis

-   -   Complete suite of image processing and quantitative analysis        functions Image stitching algorithms will allow stitching of a        series of panoramic or partially overlapping images of a wound        into a single image, either in automated or manual mode.    -   Easy to use measurement tools    -   Intuitive set up of processing parameters    -   Convenient manual editor

Report Generation

-   -   Powerful image report generator with professional templates        which may be integrated into existing clinical report        infrastructures, or telemedicine/e-health patient medical data        infrastructures. Reports may be exported to PDF, Word, Excel,        for example.

Large Library of Automated Solutions

-   -   Customized automated solutions for various areas of wound        assessment including quantitative image analysis.

Although image analysis algorithm, techniques, or software have beendescribed, this description also extends to a computing device, asystem, and a method for carrying out this image analysis.

Image-Guidance

Devices in accordance with the present disclosure may also be useful forproviding fluorescent image-guidance, for example in surgicalprocedures, even without the use of dyes or markers. Certain tissuesand/or organs may have different fluorescent spectra (e.g., endogenousfluorescence) when viewed using the imaging device, or example undercertain excitation light conditions.

FIG. 36 demonstrates the usefulness of a device in accordance with thepresent disclosure for fluorescence imaging-assisted surgery. With theaid of fluorescence imaging using the device, different organs of amouse model may be more clearly distinguishable than under white light.Insets b), c) and g) show the mouse model under white light. Insets a),d)-f), and h)-j) of FIG. 36 show the mouse model as imaged with thedevice.

FIG. 37 shows an example of the use of a device in accordance with thepresent disclosure for imaging small animal models. Here, the mousedorsal skin-fold window chamber is imaged under white light (insets a)and c) of FIG. 37 ) and fluorescence (insets b) and d) of FIG. 37 ).Note the high-resolution white light and fluorescence images obtained bythe device. The feet and face appear bright red fluorescent due toendogenous autofluorescence from the cage bedding and food dustmaterials. (405 nm excitation; 490-550 nm and >600 nm emissionchannels).

Bioengineered Skin

Several bioengineered skin products or skin equivalents have becomeavailable commercially for the treatment of acute and chronic wounds, aswell as burn wounds. These have been developed and tested in humanwounds. Skin equivalents may contain living cells, such as fibroblastsor keratinocytes, or both, while others are made of acellular materialsor extracts of living cells. The clinical effect of these constructs is15-20% better than conventional ‘control’ therapy, but there is debateover what constitutes an appropriate control. Bioengineered skin maywork by delivering living cells which are known as a ‘smart material’because they are capable of adapting to their environment. There isevidence that some of these living constructs are able to release growthfactors and cytokines. Exogenous fluorescent molecular agents may beused in conjunction with such skin substitutes to determine completenessof engraftment as well as biological response of the wound to thetherapy. The healing of full-thickness skin defects may requireextensive synthesis and remodeling of dermal and epidermal components.Fibroblasts play an important role in this process and are beingincorporated in the latest generation of artificial dermal substitutes.

The exemplary imaging devices described herein may be used to determinethe fate of fibroblasts seeded in skin substitute and the influence ofthe seeded fibroblasts on cell migration and dermal substitutedegradation after transplantation to a wound site. Wounds may be treatedwith either dermal substitutes seeded with autologous fibroblasts oracellular substitutes. Seeded fibroblasts, labeled with a fluorescentcell marker, may then be detected in the wounds with a fluorescenceimaging device and then quantitatively assessed using image analysis,for example as described above.

Polymer-Based Therapeutic Agents

There are a number of commercially available medical polymer productsmade for wound care. For example, Rimon Therapeutics produces Theramers™(www.rimontherapeutics.com) which are medical polymers that havebiological activity in and of themselves, without the use of drugs.Rimon Therapeutics produces the following wound care products, which canbe made to be uniquely fluorescent, when excited by 405 nm excitationlight: Angiogenic Theramer™, which induces new blood vessel development(i.e., angiogenesis) in wounds or other ischemic tissue; MI Theramer™,which inhibits the activity of matrix metalloproteases (MMPs), aubiquitous group of enzymes that are implicated in many conditions inwhich tissue is weakened or destroyed; AM Theramer™, a thermoplasticthat kills gram positive and gram negative bacteria without harmingmammalian cells; and ThermaGel™, a polymer that changes from a liquid toa strong gel reversibly around body temperature. These can each be madeto be fluorescent by addition of fluorescent dyes or fluorescentnanoparticles selected to be excited, for example, at 405 nm light withlonger wavelength fluorescence emission.

By using the exemplary imaging devices of the present disclosure, theapplication of such fluorescent polymer agents may be guided byfluorescent imaging in real-time. This may permit the Theramer agent tobe accurately delivered/applied (e.g., topically) to the wound site.Following application of the agent to the wound, a fluorescent imagingdevice may then be used to quantitatively determine the therapeuticeffects of the Theramers on the wound as well as track thebiodistribution of these in the wound over time, in vivo andnon-invasively. It may also be possible to add a molecular beacon,possibly having another fluorescent emission wavelength, to the MITheramer™ that can fluoresce in the presence of wound enzymes (e.g.,MMPs), and this may indicate in real-time the response of the wound tothe MI Theramer™. It may be possible to use one fluorescence emissionfor image-guided Theramer application to the wound site and anotherdifferent fluorescence emission for therapeutic response monitoring, andother fluorescence emissions for other measurements. The relativeeffectiveness of MMP inhibition and antimicrobial treatments may bedetermined simultaneously over time. Using image analysis, real-timecomparison of changes in fluorescence of these signals in the wound maybe possible. This adds a quantitative aspect to the device and adds toits clinical usefulness.

It should be noted that other custom bio-safe fluorescence agents may beadded to the following materials which are currently used for woundcare. The fluorescent material may then be imaged and monitored usingthe disclosed devices.

-   -   Moist Wound Dressings: This provides a moist conducive        environment for better healing rates as compared to traditional        dressings. The primary consumer base that manufacturers target        for these dressings is people over the age of 65 years,        suffering from chronic wounds such as pressure ulcers and venous        stasis ulcers. Those suffering from diabetes and as a result,        developed ulcers form a part of the target population.    -   Hydrogels: This adds moisture to dry wounds, creating a suitable        environment for faster healing. Their added feature is that they        may be used on infected wounds. These are also designed to dry        to lightly exudative wounds.    -   Hydrocolloid Dressings: Hydrocolloids seal the wound bed and        prevent loss of moisture. They form a gel upon absorbing        exudates to provide a moist healing environment. These are used        for light to moderately exudative wounds with no infection.    -   Alginate Dressings: These absorb wound exudates to form a gel        that provides a moist environment for healing. They are used        mainly for highly exudative wounds.    -   Foam Dressing: These absorb wound drainage and maintain a moist        wound surface, allowing an environment conducive for wound        healing. They are used on moderately exudative wounds.    -   Transparent Film Dressing: These are non-absorptive, but allow        moisture vapor permeability, thereby ensuring a moist wound        surface. They are intended for dry to lightly exudative wounds.        Examples include alginate foam transparent film dressings.    -   Antimicrobials: These provide antibacterial action to disinfect        the wound. Of particular interest is the use of nanocrystalline        silver dressings. The bio burden, particularly accumulated        proteases and toxins released by bacteria that hampers healing        and causes pain and exudation, is reduced significantly with the        extended release of silver.    -   Active Wound Dressings: These comprise highly evolved tissue        engineered products. Biomaterials and skin substitutes fall        under this category; these are composed entirely of biopolymers        such as hyaluronic acid and collagen or biopolymers in        conjunction with synthetic polymers like nylon. These dressings        actively promote wound healing by interacting either directly or        indirectly with the wound tissues. Skin substitutes are        bioengineered devices that impersonate the structure and        function of the skin.    -   Hyaluronic Acid: This is a natural component of the extra        cellular matrix and plays a significant role in the formation of        granular tissue, re-epithelialization and remodeling. It        provides hydration to the skin and acts as an absorbent.

Other wound care products that may be imaged using the disclosed devicesinclude Theramers, silver-containing gels (e.g., hydrogels), artificialskin, ADD stem cells, anti-matrix metalloproteinases, and hyaluronicacid. Fluorescent agents may be added to other products to allow forimaging using the devices. In some cases, the products may already beluminescent and may not require the addition of fluorescent agents.

The exemplary disclosed devices may be used also to monitor the effectsof such treatments over time.

Cosmetic Applications

Device in accordance with the present disclosure may be used to image apatient's skin surface. For example, the device may be used to obtainimages of the patient's skin by detecting autofluorescence produced byviolet/blue light excitation of the skin surface. Red fluorescence fromP. acnes may be easily detected in regions of the patient's face. P.acnes is the causative agent of acne vulgaris (i.e., pimples) and is acommon resident of the pilosebaceous glands of the human skin.Furthermore, P. acnes is occult under white light visualization. Theauto fluorescent images of the patient's skin may be obtained withoutthe need of exogenous agents/drugs and demonstrate the capability of thedevice to detect bacteria in single skin pores.

FIG. 39 shows an example of the use of an imaging device in accordancewith the present disclosure for real-time fluorescence detection ofcommon bacterial flora on skin. Inset a) of FIG. 39 shows redfluorescence on and around the nose detected from Propionibacteriumacnes (P. acnes) commonly found within skin pores. Inset b) shows thatfluorescence imaging may also be used to detect and monitor more thanone bacterial species on the skin at the same time, for examplePropionibacterium acne appears as red fluorescent (red arrow) whilePseudomonas Aeruginosa appears bright green (green arrows). This datasuggests the use of the device for distinguishing relativeconcentrations/levels of various bacterial species, determining theirbiodistributions on a body surface, and monitoring response toanti-bacterial treatments in dermatology and cosmetology applications.Inset c) of FIG. 39 shows an example of a fluorescence image of aculture grown on agar from a swab taken from normal skin on the nose ofa healthy volunteer. Bacteriology results showed the presence ofPseudomonas aeruginosa.

Such a capability to image and document the presence and biodistributionof bacteria on the skin surface makes the device potentially useful inthe dermatology and cosmetology fields. For example, fluorescenceimaging may be performed prior to, during, and after application ofdermatological treatment and/or pharmaceutical/cosmetic formulations(e.g., topical creams, drugs and other antibiotics, skin disinfectingagents, acne treatments, etc.) to the normal and abnormal skinconditions, including but not limited to scarring, hyper-pigmentation,acne, psoriasis, eczema, rashes, etc. Fluorescence/reflectanceimage-guided tattoo removal (e.g., using surgery or available lasertreatments) may also be an option with the device.

The device was also used to image minor cuts, scrapes, and abrasions onpatients' skin and under violet/blue light. Tissue autofluorescence fromconnective tissue components (e.g., collagen and elastin) from the woundsite and surrounding normal skin aided in detecting white light-occultchanges in connective tissues during minor cutaneous wound healing (asseen in FIG. 40 insets f) and g)). In addition, the device may alsoserve as a practical, cost-effective and sensitive image-based tool forearly detection of occult skin cancers and non-cancerous (i.e., benign)lesions in a non-invasive manner. The device may then be used to provideimage-guidance for surgical excision of the lesions or for PDT. For thelatter, fluorescence imaging may monitor PDT response and determinecompleteness of treatment over-time with multiple longitudinal imagescans of the affected area. The device may be used in real-time fordetermining PDT photosensitizer localization and biodistribution andphotobleaching, and this may be mapped onto the white light image of thearea to be treated for anatomical comparison. Changes in the opticalproperties between normal and diseases or burned tissues may be detectedusing both then white light and fluorescence imaging capabilities of thedevice.

With reference now to FIG. 40 a device in accordance with the presentdisclosure was used to image various patient skin surfaces. In insetsa)-c) of FIG. 40 , the device was used to image the skin on patients'faces by detecting autofluorescence produced by violet/blue lightexcitation of the skin surface. Red fluorescence from P. acnes mayeasily be detected in regions of the face (inset (e) of FIG. 40 ). Thedevice may be used to image and/or monitor the potential effects ofdermatological interventions (e.g., topical creams, drugs and otherantibiotics, etc.) on patients' skin. In insets d) and e) of FIG. 40 ,the device was also used to image minor cuts scrapes and abrasions onpatients' skin, as well as psoriasis on a finger. Under violet/bluelight, the device detected tissue autofluorescence from connectivetissue components (e.g., collagen and elastin) from the wound site andsurrounding normal skin to yield high-resolution images of subtlecutaneous lesions.

The devices may also be used to image, assess and longitudinally monitorthe healing process in burns or determine the response of skin grafts ortemporary skin substitutes in treatment of burn patients. The devicesmay also serve to detect and monitor late radiation-induced skin damageduring treatment of patients with ionizing radiation.

FIG. 41 shows an example of the use of a device in accordance with thepresent disclosure for imaging of cosmetic products. For example, fourcommercially available cosmetic creams are shown under white light(inset a) of FIG. 41 ) and fluorescence imaging modes (inset b) of FIG.41 ), showing fluorescence contrast between the creams and thebackground skin. This data illustrates the potential use of the handheldimaging devices for use in imaging the presence and potential biologicaleffects of cosmetic (e.g. rehydration of skin, collagen remodeling,repairing sunburn damage, skin exfoliation) and/or dermatological agentsor drugs (405 nm excitation; 490-550 nm and >600 nm emission channels)).

Darkening Environment

In accordance with various embodiments of the present disclosure, it maybe beneficial to use the disclosed imaging devices in a reduced lightingenvironment, such as a dimly lit environment or completely darkenvironment, to obtain FL images. In cases where ambient lighting cannotbe dimmed, reduced sufficiently, or completely turned off, the optimallighting conditions can be managed with optical accessories, such as atent or drape. For example, when using the device of the presentdisclosure in a lit environment (such as in an operating room), a tentor drape may be used to create a darkened (dimly lit or complete dark)environment around the imaging target, for example around a limb of apatient. In some embodiments, the device includes a mechanism to allowfor attachment of the tent or drape to the imaging device. The tent ordrape may be disposable and may be packaged with the device as part of asystem.

In some embodiments, the imaging device in accordance with the presentdisclosure may be used in a room (such as an operating room) and one ormore lights in the room may be turned off to produce the dimly lit orcompletely dark environment required for fluorescence imaging. In thisembodiment, the imaging device may also be used with the tent or drape.

In some exemplary embodiments, the device may also be configured toprompt a user to confirm that the lighting conditions in the environmentare sufficient (i.e., dimmed/darkened) when enabling the FLfunctionality of the imaging device. In other exemplary embodiments, thedevice may also display an indicator, such as, for example a moon iconon the image, to denote that the image was taken in an appropriateenvironment (i.e., in a dimmed and/or darkened environment).

FIG. 42 shows an exemplary embodiment in which an imaging device 400 isused with a drape 410 for imaging a wound 420 on a patient 430 inaccordance with the present disclosure. However, it is also contemplatedthat the device 400 and drape 410 may be used for other purposes, suchas to image a patient's skin for cosmetic purposes, as discussed above.As shown in FIG. 42 , the device 400 is connected to the drape to imagea wound in the dimly lit or completely dark environment created by thedrape 410. Thus, the drape or tent creates a darkened environment withinwhich the target may be imaged. Those of ordinary skill in the art willunderstand, however, that the drape illustrated in FIG. 42 is exemplaryonly and that various types and/or configurations of drapes and/or tentsmay be used in conjunction with the disclosed imaging devices to producethe required dimly lit and/or darkened environment, without departingfrom the present disclosure and claims.

Kits for Device

Imaging devices in accordance with the present disclosure also may beprovided in a kit, for example including the device and a fluorescingcontrast agent. The contrast agent may be any one or more of thosedescribed above. For example, the contrast agent may be for labeling abiomarker in a wound, where the kit is for wound monitoringapplications. Alternatively, the imaging device and a drape may bepackaged or otherwise provided together.

FIG. 38 shows an example of a kit including an exemplary imaging device.Inset a) of FIG. 38 shows the handle and the touch-sensitive viewingscreen, and inset b) of FIG. 38 shows an external housing and excitationlight sources. The imaging device may be used to scan the body surfaceof both human and veterinary patients for image-based wound assessment,or for non-wound imaging applications. The device and any accessories(e.g., electrical/battery power supplies), potential exogenousfluorescence contrast agents, etc.) may be conveniently placed intohard-case containers for transport within clinical and non-clinicalenvironments (including remote sites, home care and research laboratorysettings).

The imaging device may be used in white light and fluorescence modes toimprove the administration of these treatments as well as monitor theireffectiveness over time non-invasively and quantitatively. The devicemay be used in combination with other imaging modalities, for examplethermal imaging methods, among others.

While the present disclosure has been disclosed in terms of exemplaryembodiments in order to facilitate better understanding of thedisclosure, it should be appreciated that the disclosure can be embodiedin various ways without departing from the principle of the disclosure.Therefore, the disclosure should be understood to include all possibleembodiments which can be embodied without departing from the principleof the disclosure set out in the appended claims. Furthermore, althoughthe present disclosure has been discussed with relation to woundimaging, monitoring, and analysis those of ordinary skill in the artwould understand that the present teachings as disclosed would workequally well in various other applications such as, for example,clinically- and research-based imaging of small and large (e.g.,veterinary) animals; detection and monitoring of contamination (e.g.,bacterial contamination) in food/animal product preparation in the meat,poultry, dairy, fish, agricultural industries; detection of ‘surfacecontamination’ (e.g., bacterial or biological contamination) in public(e.g., health care) and private settings; multi-spectral imaging anddetection of cancers in human and/or veterinary patients; as a researchtool for multi-spectral imaging and monitoring of cancers inexperimental animal models of human diseases (e.g., wound and cancers);forensic detection, for example of latent finger prints and biologicalfluids on non-biological surfaces; imaging and monitoring of dentalplaques, carries and cancers in the oral cavity; periodontal disease;cancers in the tongue; imaging of an ear-nose-throat target, imaging ofan ocular target; imaging of a genital target; imaging of an analtarget; imaging of other suitable targets on a subject; burn wounds;imaging and monitoring device in clinical microbiology laboratories; andtesting anti-bacterial (e.g., antibiotic), disinfectant agents. The useof a fluorescent imaging device in such environments is disclosed inU.S. Pat. No. 9,042,967 B2 to DaCosta et al., entitled “Device andMethod for Wound Imaging and Monitoring,” and issued on May 26, 2015,which is incorporated by reference herein. Additionally oralternatively, the device may be used for detecting and imaging of thepresence of bacteria or microbes and other pathogens on a variety ofsurfaces, materials, instruments (e.g., surgical instruments) inhospitals, chronic care facilities, old age homes, and other health caresettings where contamination may be the leading source of infection. Thedevice may be used in conjunction with standard detection,identification and enumeration of indicator organisms and pathogensstrategies.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the written description and claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a sensor” includes two or more different sensors. As usedherein, the term “include” and its grammatical variants are intended tobe non-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or added to thelisted items.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the system and method of thepresent disclosure without departing from the scope its teachings. Otherembodiments of the disclosure will be apparent to those skilled in theart from consideration of the specification and practice of theteachings disclosed herein. It is intended that the specification andembodiment described herein be considered as exemplary only.

1. A system for determining a bacterial load of a target, comprising: anadaptor for configuring a mobile communication device for fluorescentimaging of a target, the adaptor comprising: a housing configured to beremovably coupled to a mobile communication device, and an excitationlight source configured to emit excitation light selected to elicitemission of bacterial autofluorescence from bacteria in a targetilluminated with the excitation light; and a mobile communication devicecomprising: an optical sensor configured to detect signals responsive toillumination of the target with the excitation light, and a processorconfigured to: receive the signals responsive to illumination of thetarget with the excitation light and corresponding to bacterialautofluorescence of the target, analyze the signals using pixelintensity, and output data regarding a bacterial load of the target. 2.The system of claim 1, wherein the excitation light source is configuredto emit excitation light having a wavelength of between about 400 nm andabout 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550nm, 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nmto about 700 nm, about 700 nm to about 750 nm, and combinations thereof.3. The system of claim 2, wherein the excitation light source isconfigured to emit blue/violet light.
 4. The system of claim 3, whereinthe excitation light source is configured to emit excitation lighthaving a wavelength of between about 400 nm and about 450 nm.
 5. Thesystem of claim 4, wherein the excitation light source is configured toemit excitation light having a wavelength of about 405 nm±20 nm.
 6. Thesystem of claim 1, wherein the adaptor further comprises a macro lens.7. The system of claim 1, wherein the adaptor further comprises a whitelight source configured to emit white light for illumination of thetarget during standard imaging.
 8. The system of claim 1, wherein thehousing includes an opening configured to receive a portion of a mobilecommunication device.
 9. The system of claim 8, wherein the opening isconfigured to receive the mobile communication device in a manner thataligns an optical sensor of the mobile communication device with anemission filter.
 10. The system of claim 1, further comprising anemission filter configured to block reflected excitation light.
 11. Thesystem of claim 1, further comprising an emission filter configured topermit passage of emissions having a wavelength of about 500 nm to about550 nm and/or a wavelength of about 590 nm to about 690 nm.
 12. Thesystem of claim 1, further comprising an emission filter is configuredto permit passage of emissions having a wavelength of about 450 nm toabout 505 nm and/or a wavelength of about 590 nm to about 650 nm. 13.The system of claim 1, further comprising an emission filter isconfigured to permit passage of emissions having a wavelength of about635+/−10 nm.
 14. The system of claim 11, wherein the emission filter isconfigured to move between a first position and a second position. 15.The system of claim 14, wherein, when the adaptor is removably coupledto the mobile communication device, the first position aligns theemission filter with an optical sensor of the mobile communicationdevice and the second position moves the emission filter out ofalignment with the optical sensor of the mobile communication device.16. The system of claim 1, wherein the excitation light source is afirst excitation light source configured to emit a first excitationlight at a first wavelength or wavelength band and further comprising asecond excitation light source configured to emit a second excitationlight at a second wavelength or wavelength band, wherein the firstwavelength or wavelength band is different than the second wavelength orwavelength band.
 17. The system of claim 16, wherein the firstwavelength or wavelength band is between about 400 nm and 450 nm and thesecond wavelength or wavelength band is between about 450 nm to about500 nm, about 500 nm to about 550 nm, 550 nm to about 600 nm, about 600nm to about 650 nm, about 650 nm to about 700 nm, about 700 nm to about750 nm, and combinations thereof.
 18. The system of claim 17, whereinthe first excitation light source is configured to emit excitation lighthaving a wavelength of about 405 nm±20 nm.
 19. The system of claim 16,wherein the first excitation light source is configured to emitblue/violet light and the second excitation light source is configuredto emit green light.
 20. The system of claim 19, wherein the adaptorfurther comprises a thermal sensor for capturing thermal data from thetarget.
 21. The system of claim 1, wherein the adaptor further comprisesa thermal sensor for capturing thermal data from the target.
 22. Thesystem of claim 21, wherein the processor is further configured toreceive the thermal data, correlate the thermal data with data based onthe signals responsive to illumination of the target with the excitationlight and corresponding to bacterial autofluorescence of the target, andprovide an output based on a correlation of the data.
 23. The system ofclaim 21, wherein the processor is further configured to receive thethermal data and co-register the thermal data with bacterialautofluorescence data of the target, and provide an output based on aco-registration of the data.
 24. The system of claim 23, wherein theoutput based on the co-registration of the data comprises one or more ofan image, a map, a graphic, or other visual indication of theco-registered bacterial autofluorescence data and the thermal data. 25.The system of claim 24, further comprising a display for displaying theimage, map, graphic, or other visual indication of the co-registeredbacterial autofluorescence data and the thermal data output by theprocessor.
 26. The system of claim 1, wherein the adaptor furthercomprises a power source for the excitation light source.
 27. The systemof claim 22, wherein the target is a wound in tissue and the outputbased on the correlation of the data includes one or more of anindication of wound status, an indication of wound healing, anindication of wound infection, bacterial load of the wound, temperatureof the wound, and distribution of bacteria within the wound.
 28. Thesystem of claim 1, wherein the excitation light source is configured toemit light in one of ultraviolet, visible, near-infrared, and infraredranges.